Cancer immunotherapy with semi-allogeneic cells

ABSTRACT

The present invention relates to improved semi-allogeneic immunogenic cells which act to stimulate and induce an immunological response when administered to an individual. In particular, it relates to cells which express both allogeneic and syngeneic MHC determinants and which also express at least one antigen recognized by T lymphocytes. The invention is also directed to methods of inducing an immune response and methods of treating tumors by administering the semi-allogeneic immunogenic cells to an individual.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of provisional application Ser. No.60/036,620, filed Jan. 31, 1997 (abandoned).

STATEMENT REGARDING GOVERNMENT SPONSORSHIP

This invention was made with United States government support underGrant No. RO1-CA-55651-02 awarded by the National Institutes of Health.The United States government may have certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to improved semi-allogeneic immunogeniccells which act to stimulate and induce an immunological response whenadministered to an individual. In particular, it relates to cells whichexpress both allogeneic and syngeneic MHC determinants and which alsoexpress at least one antigen recognized by T lymphocytes. The inventionis also directed to methods of inducing an immune response and methodsof treating tumors by administering the semi-allogeneic immunogeniccells to an individual.

2. State of the Prior Art

T lymphocytes recognize an extraordinarily wide array of relativelysmall peptides derived from larger macromolecules in the context ofmembrane-associated structures specified by the class-I majorhistocompatibility complex (MHC).

Most progressively growing neoplastic cells form potential immunogenictumor associated antigens (TAAs). TAAs have been identified for a numberof tumors, including melanoma, breast adenocarcioma, prostaticadenocarcinoma, esophageal cancer, lymphoma, and many others. See reviewby Shawler et al. Advance in Pharmacology 40:309-337, Academic Press(1997). Like other epitopes, TAAs on tumor cells are recognized by Tlymphocytes in the context of MHC-specified determinants. Traversari etal. (1992) J Exo. Med. 176:1453-1457; van der Bruggen et al. (1991)Science 2:1643-1647. However, such tumor cells do not provoke anti-tumorimmune responses that are capable of controlling the growth of malignantcells. Boon et al. (1992) Cancer Surveys 13:23-37; Boon, T. (1993) Int.J. Cancer 54:177-180; Boon, T. (1992) Advances Cancer Res. 58:177-209.

In recent years, attention has focused on the use of cytokines in anattempt to augment the immune response to tumor-associated antigens.Cytokines such as interleukin 2 (IL-2) or interferon (IFN-γ) have beenused to treat neoplastic disease with marginal therapeutic impact.Vieweg et al. (1995) Cancer Investigation 132(2):193-201. Cytokines donot exhibit direct toxic effect on cancer cells; their anti-tumoractivity is mediated by modulation of the host's immunological responseto the neoplasm. For example, interferon-γ induces the expression of MHCclass I determinants and augments the sensitivity of tumor cells tocytotoxic T cell-mediated lysis. Lichtor et al. (1995) J. Neurosurg83:1038-1044. IL-2 is required for the growth of cytotoxic T lymphocytesand enhances natural killer (NK) and lymphokine-activated killer cells(LAK). The limited effect of systemic administration of IL-2 in cancerimmunotherapy has been partially explained by the short half-life ofIL-2 and severe toxicity due to necessary high doses. Vieweg et al.(1995).

Lymphokine-activated killer cells (LAK) have also been used as anapproach to elicit a cellular immune response. LAK cells areMHC-unrestricted lymphoid cells which kill fresh tumor cells but notnormal cells. Tumor-infiltrating lymphocytes (TIL) are predominantlyMHC-restricted T cells which have been found to be 50-100 times morepotent than LAK cells in murine models. The use of LAK or TIL eitheralone or with IL-2 has shown some anti-tumor effects. In the combinedapproach however, IL-2 toxicity remains a problem. Vieweg et al. (1995).

More recently, immunotherapy of neoplastic disease has involved theintroduction of genes for cytokines into autologous malignant cellswhich are then introduced into immunocompetent recipients. Theintroduction and expression of the gene for IL-2 or IFN-γ into a tumorcell, usually by retroviral transduction, results in recognition of thecells by the immune system, a decrease in the cells' metastaticproperties and the generation of immune responses that are capable ofcausing the rejection of both cytokine-secreting and the originalcytokine non-secreting tumor cells. As occurs with other therapeuticstrategies, elimination of the entire neoplastic cell population isoften incomplete and tumor growth recurs. Cohen et al. (1994) Seminarsin Cancer Biology 5:419-428.

In related studies, the introduction of genes specifying defined, butallogeneic (foreign to the recipient) MHC class I determinants intomurine tumor cells leads to a loss of the cells' tumorigenicity inimmunocompetent recipients. Similar to tumor cells which have beenmodified for cytokine secretion, mice rejecting tumor cells expressingboth syngeneic and allogeneic antigens express immunity towardunmodified neoplasms expressing syngeneic determinants alone. Survivalof tumor-bearing mice immunized with the modified cells is significantlylonger than that of nonimmunized mice, although in most instances, tumorgrowth recurs and the animals eventually succumb to the disease. Itayaet al. (1987) Cancer Res. 47:3136-3140; Hui et al. (1989) J. Imunol.143:3835-43; Ostrand-Rosenberg (1991) Int. J. Cancer [Suppl] 6:61-8.

Modification of tumor cells for purposes of immunotherapy requiresestablishment of a cell line from the patient's malignant cells.Establishing such a cell line cannot always be accomplished, as is shownby, e.g., Oettgen, et al., Immunol. Allergy. Clin. North. Am. 10:607-637(1990). In addition, malignant cells isolated from a patient which arecapable of growing in vitro may not be reflective of the patient'sneoplasm as a whole. That is, tumor associated antigens present on onlya small population of cells may not be included in cells which arecapable of growing in vitro. Moreover, in those rare instances where along term malignant cell line can be established, transduction of celllines and post transduction selection can result in selective loss oftumor associated antigens expressed by the parental malignant cells invivo.

Recent studies in cancer immunotherapies have involved the use ofallogeneic cells such as mouse fibroblasts which have been geneticallyengineered to express (antibody-defined) melanoma-associated antigens(MAAs) and to secrete IL-2. Mice with established melanoma and immunizedwith the modified fibroblasts develop strong cellular anti-melanomaimmune responses, mediated primarily by CD8⁺ T-cells, macrophages andnatural killer/lymphokine-activated killer (NK/LAK) cells. Immunizedmice survive significantly longer than both nonimmunized mice and miceimmunized with irradiated melanoma cells. Kim et al. (1992) Int. J.Cancer 51:283-289.

Two nonexclusive mechanisms have been proposed to explain the improvedresponse against autologous tumors in mice immunized with allogeneiccells engineered to secrete IL-2 and express MAAs:(i) large numbers ofCTLs with specificity toward tumor-associated antigens expressed by theneoplasm are generated in the micro environment of allograft recognitionand rejection (the immunogenic properties of tumor cells transfectedwith genes specifying allogenic determinants is supportive, Hui et al.(1989) J. Immunol. 143:3835-3843; Ostrand-Rosenberg et al. (1991) J.Cancer 6:[Suppl.]:61-680); and (ii) allogeneic MHC class I determinantspresent tumor-associated T-cell epitopes directly to CTL precursors. Thehigh, local environment of IL-2, secreted by the genetically modifiedcells, further augments the generation of large numbers of CTLs withanti-tumor specificity.

Although survival of tumor-bearing mice treated with IL-2 secreting, TAAexpressing, allogeneic cells is significantly (P<0.001) longer than thatof untreated mice, in most instances the tumor cell population isincompletely eradicated and the mice eventually die from progressivemalignant melanoma. Kim et al. (1994) Cancer Immunol. Immunother.38:185-193. The state of gene therapy is generally assessed by Roth andCristiano, J. National Cancer Institute 89(1): 21-39 (1997), however,significant obstacles in cancer immunotherapy have yet to be overcome.

Accordingly, there is a need for more effective cellular immunogeniccells which elicit stronger and longer lasting T-cell mediated immuneresponses against cancerous cells in the body.

SUMMARY OF THE INVENTION

The present invention is directed to semi-allogeneic immunogenic cellsgenetically selected which express at least one class I MHC or class IIMHC determinant that is syngeneic to a recipient, at least one class Ior class II MHC determinant that is allogeneic to the recipient, and atleast one antigen recognized by T cells.

In one aspect of the invention, the semi-allogeneic immunogenic cellscomprise an antigen presenting cell expressing at least one of class Ior class II MHC determinants wherein at least one class I MHC or classII MHC determinant is syngeneic to a recipient and wherein at least oneof the class I or class II MHC determinants expressed by the antigenpresenting cell is allogeneic to the recipient, and wherein said antigenpresenting cell is transformed with and expresses nucleic acid moleculescoding for at least one antigen recognized by T cells.

In one embodiment of the invention, the nucleic acid molecules codingfor at least one antigen recognized by T cells comprise a known codingsequence for an antigen recognized by T cells. The coding sequencescontemplated by the present invention include coding sequences from aninfectious agent, such as a bacterium, virus, or parasite, as well ascoding sequences for tumor associated antigens (TAAs).

The preferred coding sequences of the present invention are those codingfor tumor associated antigens (TAAs). A number of known TAA-codingsequences may be used for such purposes, which include but are notlimited to genes of the MAGE family, BAGE, Tyrosinase, CEA, CO17-1A,MART-1, gp100, MUC-1, TAG-72, CA 125, Decapeptide 810, P1A; mutatedproto-oncogenes such as p21^(ras), P210 gene, and HER-2/neu; mutatedtumor suppressor genes such as p53; (4) tumor associated viral antigenssuch as HPV16 E7. Such genes are amply described in the literature;e.g., Shawler et al. (1997).

In another embodiment of the present invention, the nucleic acidmolecules coding for at least one antigen recognized by T cells comprisegenomic DNA or RNA isolated from an infectious agent, such as abacterium, virus or parasite, or from tumor cells. According to thepresent invention, the tumor cells used for isolating DNA or RNA mayinclude cells from a tumor cell line, as well as cells from a neoplasmor a tumor of a recipient. Many tumor cell lines are available for thispurpose, such as mouse B16 melanoma cells, mouse EO771 mammaryadenocarcinoma cells and human tumor cell lines. Preferably, the tumorcells from which the DNA or RNA is isolated are obtained from a solid ordiffuse neoplasm (i.e., solid or hematological tumor) of a recipient.The neoplasms include but are not limited to melanoma, lymphoma,plasmocytoma, sarcoma, glioma, thymoma, leukemias, breast cancer,prostate cancer, colon cancer, esophageal cancer, brain cancer, lungcancer, ovary cancer, cervical cancer, hepatoma, and other neoplasmsknown in the art, such as those described by Shawler et al. (1997).

In another aspect of the invention, the semi-allogeneic immunogeniccells comprise a semi-allogeneic hybrid cell formed by fusing an antigenpresenting cell with a tumor cell, wherein the hybrid cell expresses atleast one class I MHC or class II MHC determinant that is syngeneic to arecipient and at least one class I or class II MHC determinant that isallogeneic to the recipient, and wherein the hybrid cell also expressesat least one antigen recognized by T cells. In a preferred embodiment,the antigen expressed by the semi-allogeneic hybrid cell that isrecognized by T cells is a tumor associated antigen.

According to the present invention, all the tumor cells as describedhereinabove may be employed in such cell fusion, including cells from atumor cell line, as well as cells from a tumor of a recipient.Preferably, the tumor cells are obtained from a solid or diffuseneoplasm (i.e., solid or hematological tumor) of a recipient. Theneoplasms include but are not limited to melanoma, lymphoma,plasmocytoma, sarcoma, glioma, thymoma, leukemias, breast cancer,prostate cancer, colon cancer, esophageal cancer, brain cancer, lungcancer, ovary cancer, cervical cancer, hepatoma, and other neoplasmsknown in the art, such as those described by Shawler et al. (1997).

In a further aspect of the invention, the semi-allogeneic immunogeniccells are also transformed by and express a nucleic acid sequence codingfor at least one cytokine.

A still further aspect of the invention is directed to therapeuticcompositions comprising the subject semi-allogeneic immunogenic cells.

Another aspect of the invention provides methods for inducing animmunological response which comprises administering to an animal inneed of such response an immunologically effective amount of the subjectsemi-allogeneic immunogenic cells.

The present invention also provides methods of preventing or treating atumor in an animal which comprise administering to said animal ananti-tumor effective amount of the immunogen prepared in accordance withthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the compositionsand methods of the present invention will become better understood withregard to the following description, appended claims, and accompanyingdrawings. As used herein, the symbol“/” denotes that the relevant cellsare transfected with genomic DNA, while the symbol “x” denotes that therelevant cells are fused resulting hybrid cells. For example,“LM-IL-2K^(b)/B16” represents LM-IL-2K^(b) cells are transfected withgenomic DNA from B16 cells; “LM(TK-)×B16” represents hybrid cells formedby fusing LM(TK-) cells and B16 cells.

FIGS. 1A-1F graphically depicts immuno-fluorescent staining of LM-IL-2cells transduced with pBR327H-2K^(b) with mAbs forH-2K^(b)-determinants. The fine line indicates cells incubated withIgG_(2a) isotype serum. The bold line indicates cells incubated withanti-H-2K^(b), anti-H-2K^(K), or anti-H-2D^(b) mAbs.

FIG. 2 graphically depicts tumor growth in C57BL/6J mice injected with amixture of B16 melanoma cells and one of the following: media (solidsquares), LMZipNeo cells (open diamonds), LM-IL-2(open circles),LM-IL-2/B16 cells (filled triangles); LM-IL-2K^(b)/B16 cells (filledcircles).

FIG. 3 graphically depicts survival of C57BL/6J mice injected with amixture of B16 melanoma cells and one of the following: media (filledcircles), LM ZipNeo cells (open squares), LM-IL-2 cells (opentriangles), LM-IL-2/B16 (open circles), LM-IL-2K^(b)/B16 cells (filledtriangles).

FIG. 4 graphically depicts the time to the first appearance of tumor inmice surviving after a prior injection of B16 cells and LM-IL-2K^(b)/B16cells injected a second time with B16 cells alone. Open circlesrepresent naive mice with a mean survival time (M.S.T.) of 34.4±2.2days. Closed circles represent mice surviving 120 days and having a meansurvival time of 53.0±7.1 days.

FIG. 5 graphically depicts survival of C57BL/6J mice with melanomatreated with LM-IL-2K^(b)/B16 cells. Closed circles represent miceinjected with B16 cells alone. Open squares represent mice injected withB16 cells 20 days before LM-IL-2K^(b)/B16 cells. Open trianglesrepresent mice injected with B16 cells 10 days before LM-IL-2K^(b)/B16cells. Open circles represent mice injected with B16 cells 5 days beforeLM-IL-2K^(b)/B16 cells. Filled triangles represent mice injected with amixture of B16 cells and LM-IL-2K^(b)/B16 cells.

FIGS. 6A-6E graphically depicts cytotoxicity toward B16 cells inC57BL/6J mice injected with disrupted or intact LM-IL-2K^(b)/B16 cells.

FIG. 7 graphically depicts survival of C57BL/6J mice injected with amixture of B16 cells and non-cytokine-secreting cells. Filled circlesrepresent mice injected with B16 cells; filled squares represent miceinjected with B16 cells and LM-K^(b) cells; filled triangles representmice injected with B16 cells and LM-K^(b)/B16.

FIGS. 8A-8C depicts immunofluorescent staining of B16×LM hybrid cells. Acells incubated with anti-MAA antibodies. B cells incubated withanti-H-2K^(b) mAb. C cells incubated with anti-H-2K^(b) mAb. Dottedareas cells incubated with specific antibodies; white areas cellsincubated with IgG2a isotype serum.

FIGS. 9A-9D depicts immunofluorescent staining of B16×LM hybrid cellswith B7.1 mAb in a flow cytofluorograph. Fine lines represent cellsincubated with IgG2a isotype serum. Bold lines represent cells incubatedwith anti-B7.1 mAb.

FIG. 10 depicts survival of C57BL/6J mice injected with a mixture of B16cells and B16×LM hybrid cells. Mean survival time: (1) injected withviable B16 cells alone, 29.3±4.1 days (filled squares); (2) injectedwith viable B16 cells and irradiated B16 cells, 36.8±5.3 days (cross);(3) injected with viable B16 cells and LM(TK-) cells, 35.4±1.7 days(open circles); (4) injected with viable B16 cells, irradiated B16 cellsand LM(TK-) cells, 36.8±5.3 days (open squares); and (5) injected withviable B16 cells and B16×LM hybrid cells, 52.6±11.0 days (filledcircles). Survival of mice injected with viable B16 cells and B16±LMhybrid cells relative to survival of mice in any of the other groups,P<0.005.

FIG. 11 depicts mean survival of C57Bl/6J mice injected with a mixtureof B16×LM hybrid cells and B16 melanoma, GL 261 glioma, c1498 lymphomaor EL-4 thymoma cells. Survival of mice injected with viable B16 cellsand B16×LM hybrid cells relative to survival of mice in each of theother groups, P<0.005. Striped bars: mice injected with B16 melanoma, Gl261 glioma, c1498 lymphoma or EL-4 thymoma cells alone; dotted bars:mice injected with a mixture of B16×LM hybrid cells and one of B16melanoma, Gl 261 glioma, c1498 lymphoma and EL-4 thymoma cells.

FIG. 12 depicts cytotoxic reactions (precent of specific cytolysis)toward B16 melanoma, c1498 lymphoma, EL-4 thymoma or Gl 261 glioma cellsin mice immunized with B16×LM hybrid cells. P<0.001 for specificcytolysis of B16 cells in the presence of spleen cells from miceinjected with the hybrid cells relative to the specific cytolysis ofc1498, EL-4 or Gl 261 glioma cells. Dotted bars: spleen cells from miceinjected with B16×LM hybrid cells; striped bars: spleen cells from naiveC57BL/6 mice.

FIG. 13 depicts the effect of CD8+, CD4+ or asialo-GM1 mAb onspleen-cell mediated cytotoxic responses toward B16 cells in C57BL/6mice immunized with B16×LM hybrid cells.

FIGS. 14A-14E depicts tumor growth in C57BL/6J mice injected with amixture of EO771 breast cancer cells and LM-IL-2K^(b)/EO771 cells. Meantumor volume was derived from two dimensional measurements obtained witha dial caliper. P<0.01 for the first appearance of tumor in the group ofmice injected with EO771 cells and LM-IL-2K^(b)/EO771 cells and any ofthe other groups.

FIG. 15 depicts survival of C57BL/6J mice injected with a mixture ofEO771 breast carcinoma cells and LM-IL-2K^(b)/EO771 cells. Mean survivaltimes: Mice injected with viable EO771 cells alone, 34.5±5.8 days(filled squares); mice injected with viable EO771 cells and LM cells,41±14 days (filled triangles); mice injected with viable EO771 cells andLM-IL-2K^(b) cells, 44±9 days (open circles); mice injected with viableEO771 cells and LM-IL-2K^(b)/B16 cells, 46±11 days (filled circles); ofthe seven mice injected with viable EO771 cells and LM-IL-2K^(b)/EO771cells >110 days (filled squares); and MST for remaining mice dying fromprogressive tumor growth=54±9. P for difference in survival of miceinjected with viable EO771 cells and LM-IL-2K^(b)/EO771 cells relativeto survival of mice in each of the other groups <0.001.

FIG. 16 depicts survival of C57BL/6J mice surviving a prior injection ofEO771 cells and LM-IL-2K^(b)/EO771 cells injected with EO771 cellsalone, with filled circles representing surviving mice injected withEO771 cells and open squares representing naive mice injected with EO771cells.

FIGS. 17A-17F depicts tumor growth in C3H/HeJ mice injected with amixture of SB-1 breast cancer cells and LM-IL-2K^(b)/SB-1 cells. P<0.01for the first appearance of tumor in the group of mice injected with SP1cells and LM-IL-2K^(b)/SB-1 cells and any of the other groups.

FIG. 18 depicts survival of C3H/HeJ mice injected with a mixture of SB-1breast carcinoma cells and LM-IL-2K^(b)/SB-1 cells. Mean survival times:Mice injected with SB-1 cells alone, 29±7 days (filled squares); miceinjected with SB-1 cells and LM-IL-2 cells, 38±8 days (filledtriangles); mice injected with SB-1 cells and LM-IL-2K^(b) cells, 34±7days (open circles); mice injected with SB-1 cells and LM-IL-2/SB-1cells, 36±5 days (open triangles); mice injected with SB-1 cells andLM-IL-2K^(b)/EO771 cells, 51±18 days (open squares); mice injected withSB-1 cells and LM-IL-2K^(b)/SB-1 cells, 76±26 days (filled circles).Survival of mice injected with SB-1 cells and LM-IL-2K^(b)/SB-1 cellsrelative to survival of mice in each of the other groups p<0.01.

FIG. 19 depicts immunohistochemical staining of breast cancer in miceinjected with SB-1 cells and LM-IL-2K^(b)/SB-1 cells. Cells stainingwith CD8+ mAbs within the epithelium of the tumor are indicated by (). +indicates stromal cells lining the epithelial ducts. Horizontal bar=11.0μm.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to semi-allogeneic immunogenic cellsgenetically selected which express both allogeneic (foreign to arecipient) and syngeneic (same to the recipient) MHC determinants andalso express at least one other antigen which is recognized by Tlymphocytes of the recipient and which associates with the allogeneicand syngeneic MHC determinants. The present invention is furtherdirected to therapeutic methods employing the subject semi-allogeneicimmunogenic cells.

In accordance with the present invention, it has been surprisingly foundthat the combined expression of both syngeneic and allogeneic MHCdeterminants along with the expression of antigens recognized by Tcells, stimulates an immunological response of even greater magnitudethan immunogenic cells that express either syngeneic or allogeneic MHCdeterminants alone. For example, the combined expression of bothsyngeneic and allogeneic MHC class I determinants along with theexpression of tumor associated antigens (TAAs) in fibroblast cellsprovide highly augmented, long-term anti-tumor cellular immune responsesin mice immunized with the semi-allogeneic fibroblast cells, In someinstances, the animals reject the tumor cells and survive indefinitely.

The semi-allogeneic immunogenic cells of the present invention comprisea cell genetically selected which expresses MHC determinants that aresemi-allogeneic to a recipient and which also expresses at least oneantigen recognized by T cells.

The term “a cell” or “cells” as used herein refers to singular cells aswell as populations of cells.

The term “genetically selected” as used herein denotes cells, e.g.,antigen presenting cells, which are selected by genetic approaches toensure that such cells express MHC determinants that are semi-allogeneicand also express at least one antigen recognized by T cells. The geneticapproaches that may be employed include, but are not limited to, HLAtyping, transformation or transfection techniques for introducingnucleic acid molecules into the antigen presenting cells, and cellfusion techniques. These techniques are well known in the art and arefurther described in the disclosure which follows.

Those skilled in the art may appreciate the present invention for thesubject semi-allogeneic immunogenic cells genetically selected forimmunotherapy. Antigen presenting cells ordinarily express at least oneMHC determinant. However, antigen presenting cells ordinarily availablemay not express MHC determinants that are semi-allogeneic, i.e., thesecells may express only allogeneic MHC determinants or only syngeneicdeterminants. Such antigen presenting cells may be transformed withnucleic acid molecules encoding at least one class I or class II MHCdeterminant (either syngeneic or allogeneic) such that the transformedantigen presenting cells are selected that express both syngeneic andallogeneic determinants. In other instances, a number of donor antigenpresenting cells are available in a bank or a hospital that may or maynot express both syngeneic and allogeneic MHC determinants. According tothe present invention, (constitutive) and facultative types ofantigen-presenting cells.

It is understood that as used herein the term “fibroblast” also includesthose types of cells which develop into fibroblasts such as mesenchymalstem cells, Young et al. (1995) Dev. Dynamics 202:137-144.

In one embodiment of the present invention, the facultativeantigen-presenting cell is a fibroblast. Human fibroblast cell lines,established from normal fibroblast cells as well as malignant fibroblastcells taken from individuals, may be obtained from the American TypeCulture Collection, 12301 Parklawn Drive, Rockville, Md., 20852-1776.Human fibroblasts may also be obtained from infant foreskins aftercircumcision. Plentiful supplies of infant foreskins are available ininfant nurseries of hospitals. Macrophage cell lines and B cell linesare available through the ATCC. Other types of antigen-presenting cellsincluding fibroblasts can be isolated from tissue samples obtained fromhuman subjects.

The immunogenic cells of the present invention express MHC determinantsthat are semi-allogeneic to a recipient. “Semi-allogeneic MHCdeterminants” refers to at least one class I or class II MHC determinantexpressed by the subject immunogenic cells is syngeneic to a recipientand at least one class I or class II MHC determinant is allogeneic tothe recipient. “Syngeneic” refers to an MHC allele coding for an HLAspecificity that matches and is immunologically compatible with at leastone of appropriate donor cells are selected for immunotherapy thatexpress both allogeneic and syngeneic MHC determinants by, e.g., HLAtyping a number of donor cells and the recipient. In other instances,antigen presenting cells that are available may not express at least oneantigen recognized by T cells of the recipient. According to the presentinvention, such antigen presenting cells may then be transformed withDNA coding for at least one antigen recognized by T cells of therecipient. Antigen presenting cells may also be genetically modified by,e.g., a cell fusion process, such that the resulting hybrid cellsexpress at least one syngeneic MHC determinant, at least one allogeneicdeterminant, and at least one antigen recognized by T cells.

According to the present invention, the antigen presenting cells asreferred herein express at least one of class I or class II MHCdeterminants and may comprise those cells which are known asprofessional or constitutive antigen-presenting cells such asmacrophages, dendritic cells and B cells. Other professionalantigen-presenting cells include monocytes, macrophages, marginal zoneKupffer cells, microglia, Langerhans' cells, interdigitating dendriticcells, follicular dendritic cells, and T cells. Facultativeantigen-presenting cells may also be used in the immunogenic cells ofthe present invention. Examples of facultative antigen-presenting cellsinclude astrocytes, follicular cells, endothelium and fibroblasts. Asused herein, “antigen-presenting cells” encompass both professional aclass I or class II MHC allele of a recipient. “Allogeneic” refers to atleast one of a class I or class II MHC allele coding for an HLAspecificity that is unmatched and immunologically incompatible with atleast one of a class I or class II MHC allele of the recipient.

As described in the literature, the human MHC locus, called HLA, isfound on chromosome 6 and contains at least 50 closely-linked genes.There are three classical MHC class I genes, HLA-A, -B, and -C, each ofwhich encodes an α-chain of a MHC class I molecule. The human MHC classII genes are arranged into at least three subregions, HLA-DP, -DQ,and-DR, each of which contains at least one α gene and one β gene, Roittet al. Immunoloy, 2d ed. Gower Medical Publishing, New York, 1989.

There are a large number of genes in the MHC locus and a great degree ofpolymorphism within each MHC gene. Thus a normal human population willhave a very large number of different genotypes. Table I (taken fromRoitt et al.) lists the distinct antigenic specificities detected ateach HLA subregion. A haplotype is a set of linked MHC genes on onechromosome 6. Since an individual inherits one maternal and one paternalchromosome 6, one HLA haplotype is derived from each parent.

In mice, the MHC locus (called H-2) is found on chromosome 17. There arethree main MHC class I genes, H-2K, H-2D, and H-2L. There are also threemain MHC class II genes, H-2A, H-2E and H-2M.

The present invention describes how to genetically modify antigenpresenting cells such that these cells express MHC determinants that aresemi-allogeneic to a recipient. Under the circumstance that the antigenpresenting cell to be used expresses only allogeneic determinants, anucleic acid molecule coding for at least one syngeneic determinant maybe introduced into the antigen presenting cell by well knowntransfection or transformation procedures, such as those described bySambrook et al., 1989, Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, New York.

The working Examples describe a method of introducing a gene for a mousesyngeneic MHC class I determinant, H-2K^(b), into mouse fibroblast cellsexpressing allogeneic MHC determinants of the k haplotype, i.e.,H-2^(k). (Superscript is used to indicate a haplotype.) When employed inan animal immunotherapy regime such as human immunotherapy, the step ofintroducing a gene for a human syngeneic MHC determinant into an antigenpresenting cell may not always be necessary. Donor antigen presentingcells might be available which have at least one class I or class II MHCdeterminant that matches with one of the MHC determinants of arecipient. Such antigen presenting cells may be selected after HLAtyping the recipient and a number of possible donor antigen-presentingcells.

In accordance with the present invention, HLA typing is performed on anindividual who is to be the recipient of the subject semi-allogeneicimmunogenic cells in order to determine that individual's HLA type.Methods of HLA typing are well known to those skilled in the art, areperformed routinely in hospitals and clinical laboratories and aregenerally described in Roitt et al.(1989).

In accordance with the present invention, a bank or library may beassembled comprising different human antigen-presenting cell lines whichare maintained continuously in culture. Each antigen-presenting cellline is also HLA typed and recorded by any number of record keepingmethodologies such as a log book, computer database, etc. After HLAtyping a recipient individual, an antigen-presenting cell line is chosenfrom the library or other source so that at least one allele coding forHLA specificities expressed by the antigen-presenting cell and therecipient is unmatched. Thus, for example, with regard to MHC class Ideterminants, a recipient having an A subregion specificity of A1-A2 canreceive antigen-presenting cells having an A subregion specificity ofA28-A2. The determinants which are the product of the A2 allele in theantigen-presenting cell will match the determinants coded by the A2allele in the recipient's cells (syngeneic). In addition, at least oneallele coding for HLA specificities should also be unmatched(allogeneic) between the antigen presenting cell and the recipient. Inthis manner, both syngeneic and allogeneic determinants will be presentat the surface of the antigen-presenting cell.

In a preferred embodiment of the invention, a suitableantigen-presenting cell is chosen wherein allogeneic determinants arepredominantly expressed by the antigen-presenting cell. In thisembodiment, most alleles coding for the various HLA specificities areunmatched between the antigen-presenting cell and the recipient. Thephraseology “most alleles being unmatched at the various HLAspecificities” and the like refer to unmatched alleles between donorantigen presenting cells and recipient individual in the range of fromabout 50% to less than 100%. Similarly, as used herein, the phraseology“allogeneic determinants are predominantly expressed by theantigen-presenting cell” and the like refer to the presence ofallogeneic MHC class I or class II determinants in the range of fromabout 50% to less than 100%.

The semi-allogeneic immunogenic cells of the present invention may alsobe genetically selected by fusing an antigen presenting cell with atumor cell such that the resulting hybrid cell express both syngeneicand allogeneic MHC determinants. According to such method, an antigenpresenting cell is fused with a tumor cell via a cell fusion procedure.

According to the present invention, any antigen presenting cells asdescribed hereinabove may be used for such cell fusion. Antigenpresenting cells which are employed in cell fusion may expressexclusively allogeneic MHC determinants, or may express predominantlyallogeneic MHC determinants. The term “express exclusively allogeneicMHC determinants” refers to that the MHC determinants of the donor cellsare completely unmatched with the MHC determinants of a recipient. Theterm “express predominantly allogeneic determinants” refers to thepresence of allogeneic MHC class I or class II determinants in the rangeof from about 50% to less than 100%.

More preferably, the antigen presenting cells used for fusion arederivatives or mutant antigen presenting cells which may facilitate theselection of the resulting hybrid cells. For example, derivatives ormutant antigen presenting cells as those cells that require specialnutrition supplements or have certain drug resistances. Many suchderivatives or mutant antigen presenting cells are described andavailable in the art. For example, LM(TK-) cells, available from ATCC,are mutant fibroblast LM cells that are deficient in thymidine kinase.LM(TK-) cells die in growth medium containing HAT(hypoxanthine-aminopterin and thymidine). Those skilled in the art mayappreciate many conventional procedures for obtaining such derivativesor mutant antigen presenting cells. For example, as described in theworking examples, LM(TK-) cells may be cultured in growth mediumcontaining ouabain for a period of time such that ouabain-resistantcells are enriched in the cell population.

Tumor cells which may be used for fusion express exclusively orpredominantly syngeneic MHC determinants. The term “express exclusivelysyngeneic determinants” refers to that the MHC determinants expressed bythe donor cells are the same (matched) as the MHC determinants expressedby the recipient. The term “express predominantly syngeneicdeterminants” refers to the presence of syngeneic MHC class I or classII determinants in the range of from about 50% to less than 100%. Suchtumor cells include those cells from a tumor cell line, or morepreferably, from a recipient's neoplastic cells. Various tumor celllines are available to those skilled in the art, such as B16 melanomacells, EO771 mammary adenocarcinoma cells, EL4 thymoma cells, and humanmelanoma cell lines, all of which may be obtained from American TypeCulture Collection, Rockville, Md. (ATCC). In a preferred embodiment,the tumor cells used for fusion are from an animal, e.g. a mammal,afflicted with the tumor to be treated. Such tumors may be solid orhematological tumors, which include but are not limited to melanoma,lymphoma, plasmocytoma, sarcoma, glioma, thymoma, leukemias, breastcancer, prostate cancer, colon cancer, esophageal cancer, brain cancer,lung cancer, ovary cancer, cervical cancer, and hepatoma. Tumor cellsmay be obtained from the subject via routine clinical procedures.

Methods for cell fusion which may be employed in practicing the presentinvention are thoroughly described in the literature and includePolyethylene Glycol(or PEG) mediated-, Calcium phosphate mediated-,Lipofectin mediated- and electroporation mediated-cell fusions.

Semi-allogeneic hybrid cells resulting from a cell fusion procedure maybe selected by well-known procedures, including selections based on drugresistance or special nutrition requirements as described herein above.For example, ouabain-resistant LM(TK-) cells are resistant to ouabain,but are sensitive to HAT. B16 cells are resistant to HAT, but aresensitive to ouabain. When LM(TK-) cells are fused with B16 cells, theresulting hybrid cells are resistant to both HAT and ouabain. Suchhybrid cells may then be selected by growth medium containing bothouabain and HAT. Another example of the methods for selecting hybridcells is fluorescence-activated cell sorting (FACS), a well-knownprocedure to those skilled in the art. See, e.g., Coligan et al. CurrentProtocols in Immunology, John Wiley & Sons Inc. (1994). In this method,certain surface molecules are recognized by specific antibodies whichare fluorescently labeled. Cells which express these surface moleculesmay then be collected by a fluorescence activated cell sorter.Accordingly, hybrid cells may be selected as those cells expressingsurface molecules of both parental cells (i.e., conventional antigenpresenting cells and tumor cells) are selected as hybrid cells at theend of a cell fusion procedure. Many surface molecules may be examined,e.g., B7.1, ICAM, MHC molecules, or tumor associated antigens, againstwhich specific antibodies are available. Such hybrid cells are examinedfor their surface MHC determinants to ensure that both syngeneicdeterminants and allogeneic determinants are present at the cellsurface. Those skilled in the art may use a number of well known methodsfor this examination; for example, immunofluorescent staining followedby cytometric measurments. See Coligan et al. Current Protocols inImmunology, John Wiley & Sons Inc. (1994).

Further in accordance with the present invention, the geneticallyselecteded immunogenic cells express, in addition to semi-allogeneic MHCdeterminants, at least one antigen recognized by T cells.

In one aspect of the invention, the antigen presenting cells aregenetically transformed with nucleic acid molecules coding for at leastone antigen recognized by T cells.

In one embodiment according to this aspect of the present invention, thenucleic acid molecules coding for at least one antigen recognized by Tcells are known RNA or DNA sequences coding for at least one antigenrecognized by T cells.

Coding sequences useful for practicing the present invention maycomprise any of a myriad of known sequences or fragment of knownsequences which encode antigens recognized by T cells. “Fragment” ismeant segment of DNA having sufficient length to encode an antigenicpeptide of at least about 8 amino acids.

The present invention contemplates coding sequences for a number oftumor associated antigens, which include but are not limited to (1)genes coding for TAAs which are recognized by cellular immune responses(mediated primarily by cytotoxic T cells) and/or by humoral immuneresponses (mediated primarily by T helper cells), such as members ofMAGE gene family, BAGE, Tyrosinase, CEA, CO17-1A, MART-1, gp100, MUC-1,TAG-72, CA 125, Decapeptide 810, P1A; (2) mutated proto-oncogenes suchas p21^(ras), P210 gene (a product of bcr/abl rearrangement), andHER-2/neu; (3) mutated tumor suppressor genes such as p53; (4) tumorassociated viral antigens such as HPV E7. Genes for such TAAs are fullydescribed in the art, e.g., Shawler et al.(1997). Some tumor associatedantigens are expressed in certain types of tumors, others are associatedwith a variety of types of tumors. In accordance with the presentinvention, the skilled artisan may choose particular coding sequencesaccording to the type of tumor to be treated.

Coding sequences for antigens of an infectious agent are alsocontemplated by the present invention. The semi-allogeneic immunogeniccells of the present invention are especially useful against viruses,many of which mutate and change their outer envelope thereby frustratingneutralization by antibodies. In addition, the subject semi-allogeneicimmunogenic cells are also useful against pathogens which quickly entera host's cells and hide from circulating cells of the immune system.Examples of such intracellular parasites against which thesemi-allogeneic immunogenic cells of the present invention are usefulinclude Borrelia, Chlamydia, Plasmodium, Legionella pneumophila,Leishmania, the trypanosome responsible for Chagas' and the like.

According to the present invention, a coding sequence for an antigen isplaced in a vector which can replicate within a cell. The codingsequence is operably linked to a promoter which functions in cells of ananimal such as a mammal, and is contained within the vector. Therecombinant vector comprising the promoter and coding sequence is thenintroduced into the antigen-presenting cell. The introduction of DNAinto antigen-presenting cells can be accomplished through various wellknown procedures such as by transfection of viral and retroviral vectorscomprising the DNA, transduction into a cell of modified virusparticles, and physical/chemical techniques such as calcium phosphatetransfection, complex formation with polycations or lipids,electroporation, particle bombardment and microinjection into nuclei.

Preferably, a selectable marker and termination sequence is included inthe recombinant vector. Polyadenylation signals may also be incorporatedinto the expression vector. Plasmid and viral vectors useful forpracticing the present invention are well known in the art and aredescribed in Sambrook et al. A Laboratory Manual, Cold Spring HarborLaboratory, N.Y. (1989). Promoters, 3′ termination sequences,polyadenylation signals and selectable marker genes which function inhuman cells are also well known in the art and discussed in Sambrook etal. (1989).

In another embodiment of the invention, the nucleic acid moleculescoding for at least one antigen recognized by T cells are DNA or RNAisolated from an infectious agent, such as a bacterium or virus, or fromtumor cells. Such DNA or RNA is isolated and mechanically sheared (orcut with one or more appropriate restriction enzymes in the case ofDNA), in order to generate high molecular weight fragments. The highmolecular weight fragments are then introduced into the subjectantigen-presenting cell. Virus particles may also be directly introducedinto the antigen-presenting cell by transduction.

In a more preferred embodiment, genomic DNA is isolated from tumorcells, either from a tumor cell line as described hereinabove, or morepreferably, from an animal's small primary or metastatic neoplasms, fortransfer into the antigen presenting cells.

Tumor cells taken from a subject may be used directly for isolating DNAwithout further culturing in vitro. The population of transfected cells,selected for their general, nonspecific, immune-augmenting properties,expresses the range of tumor associated antigens that characterize ananimal's tumor, including antigens that may be present on only a smallproportion of the malignant cells.

In this aspect of the invention, neoplastic cells from either diffuseneoplasms or from part of an animal's tumor, are obtained duringsurgery, by needle aspiration or other well-known methods. Examples ofneoplastic disease amenable to the practice of the present inventioninclude solid tumors and hematological tumors, e.g., melanoma, lymphoma,plasmocytoma, sarcoma, glioma, thymoma, leukemias, breast cancer,prostate cancer, colon cancer, esophageal cancer, brain cancer, lungcancer, ovary cancer, cervical cancer, and hepatoma. Genomic DNA is thenisolated and purified from the cell or tumor sample using methods wellknown in the art including those set out in Sambrook et al., 1989 ALaboratory Manual, Cold Spring Harbor Laboratory, N.Y. Neoplastic cellsand tumor samples may also be grown in culture using methods well knownin the art in order to increase the amount of DNA available forisolation.

Isolated, purified genomic DNA isolated from a tumor or cell culture isthen is preferably mechanically sheared or cut with an appropriaterestriction enzyme to render high molecular weight DNA fragments ofabout 20-25 Kb. In a most preferred method, the DNA is mechanicallysheared using a 23 or 25 gauge needle.

Genomic DNA may be introduced into the antigen presenting cells by anyof a number of methods known in the art such as calcium-phosphateco-precipitation, electroporation, cationic liposome-mediatedtransfection or by any of a number of other well known methods forintroducing DNA into cells. According to the present invention, genomicDNA is introduced along with a selectable marker such as a geneconferring resistance to hygromycin, neomycin or any other antibiotics.A procedure such as these is well-known in the art as co-transfection orco-transformation. transformation. See Sambrook et al. Such marker genesare usually contained in a plasmid. Plasmids such as these arewell-known and are available to those skilled in the art. In theco-transformation procedure of the present invention, the amount of theplasmid is preferably less than the amount of the genomic DNA tofacilitate the selection of tranformants. Preferably, the ratio ofplasmid vs genomic is about 1:3 to about 1:20; more preferably, theratio is about 1:10. After transformation, transformants (i.e., cellshaving received the genomic DNA and the marker gene) are selected asthose cells growing in selection medium, e.g., medium containgantibiotics.

In another aspect of the invention with regard to the subjectsemi-allogeneic immunogenic cells expressing at least one antigenrecognized by T cells, antigen presenting cells are genetically modifiedto express at least one antigen recognized by T cells via the cellfusion process as described hereinabove. Accordingly, antigen presentingcells are fused with tumor cells. The hybrid cells resulting from thefusion process express at least one T-cell recognizable antigen which isexpressed by the parental tumor cells, preferably, a tumor associatedantigen. The expression of at least one antigen on the hybrid cells maybe confirmed by a number of well-known methods, such asimmuno-fluorescent staining and cytometric measurements as describedhereinabove. In this regime, transformation or transfection of nucleicacid molecules coding for tumor associated antigens is not necessary.

As discussed hereinabove, antigen presenting cells, which express MHCdeterminants that are semi-allogeneic to a recipient and also express atleast one antigen recognized by T cells, are genetically selected via,e.g., transformation/transfection, HLA typing or cell fusion.

In another aspect of the present invention, the antigen presenting cellsemployed in the present invention produce costimulatory moleculesinvolved in T cell activation such as B7 and ICAM. For example, humanfibroblasts are known to produce costimulatory molecules such as B7-1and ICAM.

According to the present invention, the subject semi-allogeneicimmunogenic cells do not require transformion and/or expression of anucleic acid sequence coding for at least a cytokine. In a preferredembodiment of the present invention, the semi-allogeneic immunogeniccells may be engineered to express a coding sequence for at least onecytokine. In a preferred embodiment, the coding sequence is introducedinto antigen-presenting cells prior to introducing high molecular weightDNA, or an expression vector with coding sequence for a particularantigen, or a cell fusion procedure. The introduction into theantigen-presenting cells of multiple coding sequences for differentcytokines is also contemplated by the present invention. Examples ofcytokines useful for practice of the present invention includeinterleukin-1, interleukin-2, interleukin-3, interleukin-4,interleukin-5, interleukin-6, interleukin-7, interleukin-8,interleukin-9, interleukin-10, interleukin-11, interleukin-12,interferon-α, interferon-γ, tumor necrosis factor, granulocytemacrophage colony stimulating factor, and granulocyte colony stimulatingfactor.

Coding sequence for one or more cytokines may be introduced into thesemi-allogeneic antigen-presenting cell via an expression vector such asa plasmid or viral vector. Using vector construction methodologies wellknown in the art, coding sequence for at least one cytokine is operablylinked to and under the control of a promoter which functions in humancells. For example, a plasmid based vector comprising the SV40 promotermay be used. Viral vectors made from the Moloney Murine Leukemia (MoMLV)virus, adeno-virus, Herpes-virus, pox-virus and Adeno-associated virus(AAV) are useful for expressing cytokine genes in the semi-allogeneicantigen-presenting cells of the present invention. Such vectors are wellknown in the art and available through the ATCC. In one embodiment ofthe invention, the vector is pZipNeoSVIL2 which comprises a gene forhuman IL-2 and a neo^(r) gene, both under control of the Moloney MurineLeukemia virus long terminal repeat.

The present invention further provides a therapeutic compositioncomprising the semi-allogeneic immunogenic cells and a therapeuticallyacceptable carrier. As used herein, a therapeutically acceptable carrierincludes any and all solvents, including water, dispersion media,culture from cell media, isotonic agents and the like that are non-toxicto the host. Preferably, it is an aqueous isotonic buffered solutionwith a pH of around 7.0. The use of such media and agents in therapeuticcompositions is well known in the art. Except insofar as anyconventional media or agent is incompatible with the semi-allogeneicimmunogenic cells of the present invention, use of such conventionalmedia or agent in the therapeutic compositions is contemplated.Supplementary active ingredients can also be incorporated into thecompositions.

The therapeutic compositions of the present invention may beadministered to an animal in need thereof. Accordingly, the presentinvention provides methods for inducing an immune response in an animalin need of such response, which comprise administering to an animal animmunologically effective amount of the subject semi-allogeneicimmunogenic cells. The present invention also provides methods forpreventing or treating a tumor in an animal, which compriseadministering to an animal an anti-tumor effective amount of the subjectsemi-allogeneic immunogenic cells.

The term “animal” used herein encompasses all mammals, including human.Preferably, the animal of the present invention is a human subject.

The tumors contemplated by the present invention, against which theimmune response is induced, or which is to be prevented or treated, mayinclude and are not limited to melanoma, lymphoma, plasmocytoma,sarcoma, glioma, thymoma, leukemias, breast cancer, prostate cancer,colon cancer, esophageal cancer, brain cancer, lung cancer, ovarycancer, cervical cancer, hepatoma, and other neoplasms known in the art,such as those described by Shawler et al. (1997).

The immune response induced in the animal by administering the subjectsemi-allogeneic immunogens may include cellular immune responsesmediated primarily by cytotoxic T cells, capable of killing tumor cells,as well as humoral immune repsonses mediated primarily by helper Tcells, capable of activating B cells thus leading to antibodyproduction. A variety of techniques may be used for analyzing the typeof immune responses induced by the subject immunogenic cells, which arewell described in the art; e.g., Coligan et al. Current Protocols inImmunology, John Wiley & Sons Inc. (1994).

The term “preventing a tumor” used herein means the occurrence of thetumor is prevented or the onset of the tumor is significantly delayed.The term “treating a tumor” used herein means that the tumor growth issignificantly inhibited, which is reflected by, e.g., the tumor volume.Tumor volume may be determined by various known procedures, e.g.,obtaining two dimensional measurements with a dial caliper.

When “an immunologically effective amount”, “an anti-tumor effectiveamount”, or “an tumor-inhibiting effective amount” is indicated, theprecise amount of the semi-allogeneic immunogenic cells to beadministered can be determined by a physician with consideration ofindividual differences in age, weight, tumor size, extent of infectionor metastasis, and condition of the patient. It can generally be statedthat a therapeutic composition comprising the subject semi-allogeneicimmunogenic cells should be preferably administered in an amount of atleast about 1×10³ to about 5×10⁹ cells per dose.

The administration of the subject therapeutic compositions may becarried out in any convenient manner, including by aerosol inhalation,injection, ingestion, transfusion, implantation or transplantation.Preferably, the semi-allogeneic immunogens of the present invention areadministered to a patient by subcutaneous (s.c.), intraperitoneal(i.p.), intra-arterial (i.a.), or intravenous (i.v.) injection. Thetherapeutically acceptable carrier should be sterilized by techniquesknown to those skilled in the art.

The teachings of the publications cited throughout the presentspecification are herein incorporated by reference.

The invention is further illustrated by the following specific exampleswhich are not intended in any way to limit the scope of the invention.

EXAMPLE 1 Experimental Materials

Experimental Animals

Six to 8 week old specific pathogen-free C57BL/6J mice (H-2^(b)) andC3H/HeJ mice (H-2^(k)) were obtained from the Jackson Laboratory (BarHarbor, Me.). They were maintained in the animal care facilities of theUniversity of Illinois at Chicago according to NIH Guidelines for theCare and Use of Laboratory Animals. The mice were fed Purina mouse chowand water ad libitum. They were 8 to 12 weeks old when used in theexperiments.

Cell Lines

Tumor cells used in the examples were obtained and maintained asfollows. B16 cells, a highly malignant melanoma cell line derived from aspontaneous neoplasm occurring in a C57BL/6 mouse, were obtained from I.Fidler, (M. D. Anderson Cancer Center, Houston, Tex.). The cells weremaintained by serial passage in histocompatible C57BL/6J mice, or at 37°C. in a humidified 7% CO₂/air atmosphere in growth medium. C1498 cells,a spontaneously occurring lymphoma cell line of C57BL/6 mouse origin,were obtained from the American Type Culture Collection (Rockville,Md.). E1-4 thymoma cells and G1 126 glioma cells were also obtained fromthe American Type Culture Collection (Rockville, Md.). C1498 cells, E1-4thymoma cells and G1 126 glioma cells were maintained at 37° C. in ahumidified 7% CO₂/air atmosphere in growth medium. EO771 cells, amammary adenocarcinoma cell line derived from a C57BL/6 mouse, were fromthe Tumor Repository of the Division of Cancer Treatment, Diagnosis andCenters of the National Cancer Institute (Frederick, Md.). EO771 cellswere maintained by serial passage in compatible C57BL/6J mice. SB-1cells were obtained from a spontaneous breast neoplasm arising in aC3H/HeJ mouse maintained in the animal facility at the University ofIllinois at Chicago.

The antigen presenting cells used in the examples were obtained andmaintained as follows. LM cells, a fibroblast cell line derived from aC3H/HeJ mouse (H-2^(k)), were obtained from the American Type CultureCollection (Rockville, Md.). LM cells were maintained at 37° C. in ahumidified 7% CO₂/air atmosphere in growth medium. LM(TK−) cells, athymidine-kinase-deficient fibroblast cell line of a C3H/He mouse(H-2^(k)) origin, were obtained from the American Type CultureCollection (Rockville, Md.). LM(TK-) cells were maintained at 37° C. ina humidified 7% CO₂/air mixture in growth medium. Because LM(TK−) cellswere deficient in the enzyme thymidine kinase, they died within 14 daysin growth medium containing 100 μM hypoxanthine, 0.4 μM aminopterin and16 μM thymidine (HAT).

WR19M.1 cells, a mouse monocyte/macrophage cell line, were obtained fromthe American Type Culture Collection (Rockville, Md.), and weremaintained at 37° C. in a humidified 7% CO₂/air atmosphere in growthmedium Antisera.

An antiserum reactive with B16 melanoma cells was raised in C57BL/6Jmice injected intraperitoneally (i.p.) with killed (by three rounds offreezing and thawing) B16 cells suspended in Freund's complete adjuvant(Spex Industries, Inc., Metuchen, N.J.). The antiserum reacted with B16cells, but not with a variety of organs and tissues from C57BL/6J mice,or with a panel of various neoplastic cell lines (11). H-2K^(b) (cloneAF6-88.5 of BALB/c origin, IgG2a) and H-2K^(k) (clone 25-9-3 of C3Horigin, IgM) monoclonal antibodies (mAbs) were from Pharmingen, (SanDiego, Calif.). Fluoroscein isothiocyanate (FITC)-conjugated goatanti-mouse IgG was from Sigma, (St. Louis, Mo.). Anti L3T4 (CD4) mAbswere from Pharmingen (San Diego, Calif.); Lyt-2.2 (CD8) (hybridoma3.155) mAbs were from M. Mokyr, (University of Illinois, Chicago, Ill.)and anti-asialo GM1 mAbs were from Wako Chemical Co. (Dallas, Tex.).FITC labeled B7.1 mAbs were obtained from Pharmingen.

EXAMPLE 2 Modification of LM Cells for the Secretion of IL-2

LM cells (H-2^(k)), a fibroblast cell-line of C3H/HeJ mouse origin, weremodified for IL-2-secretion by transduction of the replication-defectiveretroviral vector, pZipNeoSVIL-2, using techniques described previouslyin Sugden et al. (1985) Mol. Cell. Biol. 5: 410-413. The vectorpZipNeoSVIL-2 was obtained from M. K. L. Collins, Institute of CancerResearch, London, England. The vector, packaged in GP+env AM12 cells,(from A. Bank, Columbia University, New York, N.Y.) included a gene forhuman IL-2 and a neo^(r) gene, both under control of the Moloneyleukemia virus long terminal repeat. For use as a control, LM cells weretransduced with the retroviral vector pZipNeoSV(X) (from M. K. L.Collins), also packaged in GP+env AM12 cells (LM-ZipNeo cells).pzipNeoSV(X) specified the neo^(r) gene, but lacked the gene for IL-2.

Virus-containing supernatants of GP+env AM12 cells transfected withpZipNeoSVIL-2 or pzipNeoSV(X) were added to LM cells, followed byovernight incubation at 37° C. in growth medium to which polybrene(Sigma; 5 μg/ml, final concentration) had been added. The growth mediumconsisted of Dulbecro's modified eagle's medium (DMEM) supplemented with10% fetal bovine serum (FBS) and antibiotics. The cells were maintainedfor 14 days in growth medium containing 400 μg/ml of the neomycinanalog, G418. The growth medium consisted of Dulbecco's modified Eagle'smedium (DMEM) supplemented with 10% fetal bovine serum (FBS). Onehundred percent of nontransduced LM cells died in the mediumsupplemented with G418 within this period. After selection, thesurviving colonies were pooled and assayed for IL-2-secretion.

IL-2 secretion was detected by the capacity of the cell culturesupernatants to sustain the growth of CTLL-2 cells, an IL-2dependentcell line (from A. Finneagan, Rush Medical College, Chicago, Ill.),Bakker et al. (1994) J. Exp. Med. 179:1005-1009. Varying dilutions ofthe filtered culture supernatants (0.2 μm nitrocellulose; Gelman, AnnArbor, Mich.) were transferred to 96-well plates (Falcon) containing5×10³ CTLL-2 cells in a final volume of 200 μl of growth medium perwell. After incubation for 16 hrs., 0.5 μCi ³H-thymidine (Amersham,Arlington Heights, Ill.) was added to each well for 6 additional hrs. Astandard curve was generated by adding varying amounts of recombinanthuman IL-2 (Gibco BRL, Grand Island, N.Y.) to an equivalent number ofCTLL-2 cells. Afterward, the cells were collected onto glass fiberfilters (Whittaker M.A. Products, Walkerville, Md.) using a PhD multipleharvester (Microbiological Associates, Bethesda, Md.) and washed withethanol (95%) Radioactivity in the insoluble fraction was measured in aliquid scintillation spectrometer (Parkard Instrument Co, Downers Grove,Ill.). One unit of IL-2 gave half maximal proliferation of CTLL-2 cellsunder these conditions. Every third transfer, the transduced cells(LM-IL-2 and LM-ZipNeo cells) were passaged in growth medium containing400 μg/ml G418.

The results (Table II) indicate that 1×10⁶ retrovirally transduced LMcells formed approximately 100 units IL-2 in 48 hrs. (LM-IL-2 cells).The culture supernatants of non transduced LM cells or LM cellstransduced with the IL-2-negative vector, pZipNeoSV(X), (LM-ZipNeocells) did not stimulate the proliferation of CTLL-2 cells. Equivalentquantities of IL-2 were detected in the culture supernatants of LM-IL-2,but not LM-ZipNeo cells for more than 6 months of continuous culture.

EXAMPLE 3 Modification of LM-IL-2 Cells for the Expression of H-2K^(b)

pBR327H-2K^(b) (Biogen Research Corp., Cambridge, Mass.), a plasmidencoding MHC H-2K^(b) -determinants was used to modify LM cells(LM-IL-2K^(b) cells). Ten μg of pBR327H-2K^(b) and 1 μg of pBabePuro (M.K. L. Collins) a plasmid conferring resistance to puromycin were mixedwith Lipofectin (Gibco BRL) according to the instructions of thesupplier, and then added to 1×10⁶ LM-IL-2 cells in 10 ml of Dulbecco'smodified Eagle's medium (DMEM) without fetal bovine serum (FBS). Theplasmid pBabePuro, Vile et al. (1993) Cancer Res. 53:962-967, wasincluded to increase the likelihood that cells that were converted toresistance to puromycin had taken up pBR327H-2K^(b). (The ratio ofpBR327H-2K^(b) to pBabePuro added to the cells was 10:1). For use as acontrol, an equivalent number of LM-IL-2 cells was transfected with 1 μgof pBabePuro alone. The cells were incubated for 18 hrs. at 37° C. in aCO₂/air atmosphere, washed with DMEM, followed by the addition of growthmedium. After incubation for 48 hrs., the cell cultures were divided andreplated in growth medium supplemented with 3.0 μg/ml puromycin (Sigma)followed by incubation at 37° C. for 7 additional days. The survivingcolonies were pooled and tested by staining with specific fluoresceinconjugated antibodies for the expression of H-2K^(b)-determinants. Onehundred percent of non-transfected LM-IL-2 cells maintained in growthmedium containing puromycin died during the seven day period ofincubation. LM-IL-2 cells transduced with the plasmid (pBR327H-2K^(b)cells), or nontransduced LM-IL-2 cells, were dissociated from 100 mm²tissue culture petri dishes with EDTA (0.1 mM) and then incubated for 1hr. at 4° with FITC-conjugated anti-H-2K^(b), anti-H-2K^(k), oranti-H2D^(b) mAbs. As a control, aliquots the cell suspensions weretreated in the same way except that FITC-conjugated-IgG_(2a) isotypeserum was substituted for the mAbs. After three washes with PBS (pH7.4), at least 1×10⁴ cells of each type were analyzed for fluorescentstaining in a flow cytofluorograph.

EXAMPLE 4 Immunofluorescent Staining and Cytofluorometric Measurments

Quantitative immunofluorescence measurements were used to detect theexpression of H-2K^(b)-determinants by LM-IL-2 cells transfected withpBR327H-2K^(b) (LM-IL-2K^(b) cells). The measurements were performed inan Epic V flow cytofluorograph (Coulter Electronics, Hialeah, Fla.)equipped with a multiparameter data-acquisition and display system(MDADS). For the analysis, a sinlge cell suspension was prepared fromthe monolayer cultures with 0.1 mM ethylene diamine tetra acetic acid(EDTA) in phosphate buffered saline (pH7.4) (PBS). The cells were washedwith PBS containing 0.2% sodium azide and 0.5% FBS. Afterward,fluorescein isothiocyanate (FITC)-conjugated H-2K^(b) monoclonalantibodies (mAbs) (clone AF6-88.5; Pharmingen, San Diego, Calif.) wereadded to the cells, followed by incubation at 4° C. for 1 hr. The cellswere then washed with PBS containing 0.5% FBS and 0.2% sodium azide.One-parameter fluorescence histograms were generated by analyzing atleast 1×10⁴ cells. Background staining was determined by substitutingcells stained with FITC-conjugated goat anti-mouse isotype IgG_(2a)alone for cells stained with the specific antibodies. The 15 percent ofcells that stained with the highest intensity were separated intoplastic cell culture plates (Falcon) containing DMEM supplemented with50% FBS. Immediately afterward, the cells were centrifuged at low speedand resuspended in growth medium in plastic cell culture plates,followed by incubation at 37° in a humidified 7% Co₂/air atmosphere. Ascontrols, aliquots of the cell-suspension, incubated with FITC-labeledIgG_(2a) isotype serum, or with FITC-labeled mAbs for H-2K^(k), orH2D^(b) determinants, were analyzed as well. The results (FIG. 1)indicated that the transfected cells (LM-IL-2K^(b) cells) stainedpositively with H-2K^(b) but not with IgG_(2a) isotype serum or H2D^(b)mAbs. As an additional control, the cells were analyzed in the same wayfor the expression of H-2K^(k) determinants. As indicated (FIG. 1),LM-IL-2 cells, of C3H/He mouse origin, stained with H-2K^(k) mAbs aswell. The intensity of immunofluorescent staining of Lm-IL-2Kb cells forH-2K^(b)-determinants was equivalent to that of spleen cells from naiveC57BL/6J mice. The expression of H-2K^(b)-determinants was a stableproperty of the cells. Cells transfected with pBR327h-2K^(b) stainedwith equivalent intensity with H-2K^(b) mAbs after three months ofcontinuous culture.

EXAMPLE 5 Transfection of LM-IL-2K^(b) Cells with Genomic DNA from B16Melanoma Cells

High molecular weight DNA isolated from B16 cells was used for thetransfection of LM-IL-2K^(b) cells, using the method described by Wigleret al., (1978) Cell 14: 725-731, as modified by Kim et al. (1992) Int.J. Cancer 51: 283-289. The DNA was first sheared by passage through anumber 25 gauge needle. The molecular size of DNA at this point wasgreater than 23 kb, as determined by electrophoreses in 0.6% agarosegels. Afterwards, 100 ug of the sheared DNA was mixed with 10 μg pHyg(from L. Lau, University of Illinois, Chicago, Ill.), a plasmid thatencodes the E. coli enzyme hygromycin B phosphotransferase (Sugden etal. (1985) Mol. Cell. Biol 5:410-413), conferring resistance tohygromycin B. The ratio of DNA of B16 cells to pHyg was 10:1 to increasethe likelihood that cells that took up the plasmid DNA also took up DNAfrom the melanoma cells as well. The sheared DNA and pHyg were thenmixed with Lipofectin, according to the manufacturer's instructions. TheDNA/Lipofectin mixture was added to a population of 1×10⁷ LM-IL-2K^(b)cells that had been divided into ten 100 mm plastic cells culture plates24 hrs previously. Immediately after adding the DNA/Lipofectin mixtureto the cells, the growth medium was replaced with DMEM. In someinstances, DNA from B16 cells was omitted and 1 ug of pHyg mixed withLipofectin was added to an equivalent number of LM-IL-2K^(b) cells. Inboth instances, the cells were maintained for 14 days in growth mediumcontaining 500 ug/ml hygromycin B (Boehringer Mannheim, Indianapolis,Ind.). One hundred percent of nontransfected LM-IL-2K^(b) cellsmaintained in the hygromycin-growth medium died within this period. Thesurviving colonies (more than 2×10⁴ in each instance) ofhygromycin-resistant LM-IL-2K^(b) cells transfected with pHyg and DNAfrom the melanoma cells (LM-IL-2K^(b)/B16) or with pHyg alone(LM-IL-2K^(b)) were pooled and used to induce an immunogenic response inmice. In some instances, the cells were disrupted by homogenization andsonication before injection into mice. The amount of IL-2 formed byLM-IL-2K^(b)/B16 cells was equivalent to that formed by LM-IL-2 orLM-IL-2K^(b) cells as determined by the capacity of the cell culturesupernatant to sustain the growth of CTLL-2 cells (Table II).

EXAMPLE 6 Survival of C57BL/6J Mice Injected with B16 Melanoma Cells andLM-IL-2K^(b) cells Transfected with Genomic DNA from B16 Cells

B16 melanoma is a highly malignant neoplasm of C57BL/6 mice. The animalsexhibited no apparent resistance to the growth of the melanoma cells.One hundred percent of mice injected subcutaneously (s.c.) with 5×10³B16 cells died from progressive tumor growth in approximately 38 days.

As a first means of determining the immunotherapeutic properties ofLM-IL-2K^(b)/B16 cells (LM-IL-2k^(b) cells transfected with genomic DNAfrom B16 cells) toward the growth of the melanoma, C57BL/6J mice wereinjected s.c. with a mixture of B16 cells and LM-IL-2K^(b)/B16 cells,followed by two subsequent injections at weekly intervals ofLM-IL-2K^(b)/B16 cells alone.

Tumor growth was monitored in C57Bl/6J mice injected with a mixture ofB16 cells and LM-IL-2K^(b)/B16 cells (FIG. 2). C57BL/6J mice (5 pergroup) were injected s.c. with a mixture of 5×10³ B16 cells and2×10⁶LM-IL-2K^(b)/B16 cells. At the same time, the mice received asecond injection i.p. of 2×10⁶ LM-IL-2 K^(b)/B16 cells alone. Ascontrols, the mice were injected according to the same protocol withequivalent numbers of B16 cells and LM-IL-2 cells, or with B16 cells andLM-IL-2/B16 cells. The mice were injected s.c. and i.p. twice more, atweekly intervals, with the same number of cells as in the initialinjections, but without the additional B16 cells. Tumor volume wasderived from two dimensional measurements obtained with a dial caliper.The results indicate that none of the mice injected with the mixture ofB16 and LM-IL-2K^(b)/B16 cells developed tumors, while mice in variouscontrol groups all developed tumors within 15-60 days (FIG. 2).

In a related experiment, the survival time of C57BL/6J mice injectedwith different combination of cell mixtures was measured (FIG. 3).C57BL/6J mice (5 per group) were injected s.c. with a mixture of5×10³B16 cells and 2×10⁶ LM-IL-2K^(b)/B16 cells. At the same time, themice received a second injection i.p. of 2×10⁶ LM-IL-2 K^(b)/B16 cellsalone. As controls, other naive (untreated) C57BL/6J mice were injectedaccording to the same protocol with equivalent number of B16 cells andLM-ZipNeo cells, with B16 cells and LM-IL-2 cells, with B16 cells andLM-IL-2/B16 cells or with B16 cells alone. The mice in each treatmentgroup were injected twice more, at weekly intervals, with the samenumber of LM-IL-2K^(b)/B16, LM-ZipNeo cells, LM-IL-2 or LM-IL-2/B16cells, but without additional B16 cells. Mean survival times were38.4+2.8 days for mice injected with viable B16 cells alone; 39.4+7.1days for mice injected with viable B16 cells and LM-ZipNeo cells,47.7+9.6 days for mice injected with viable B16 cells and LM-IL-2 cells;62.2+12.2 days for mice injected with viable B16 cells and LM-IL-2/B16cells; and >120 days for mice injected with viable B16 cells andLM-IL-2K^(b)/B16 cells. Mice injected with a mixture of B16 cells andnon IL-2secreting LM-ZipNeo cells, or with B16 cells alone, developedprogressively growing neoplasms and died in approximately 40 days. Othernaive C57BL/6J mice were injected with a mixture of B16 cells andLM-IL-2 cells. The difference in the period of survival of mice injectedwith B16 cells alone, and with the mixture of B16 cells and LM-IL-2cells was not significant (P>0.1).

To determine the involvement of H-2K^(b) -determinants in theimmunogenic properties of the cells, the survival of mice injected withB16 cells and LM-IL-2/B16 cells (LM-IL-2 cells transfected with DNA fromB16 cells) was compared to the survival of mice injected with B16 cellsand LM-IL-2K^(b)/B16 cells. The Student t test was used to determine thestatistical differences between the survival and cytotoxic activities inmice in various experimental and control groups. A p value of less than0.05 was considered significant. As shown by the data (FIG. 3), thesurvival of mice injected with the mixture of B16 cells and LM-IL-2/B16cells was significantly less than the survival of mice injected with themixture of B16 cells and LM-IL-2K^(b)/B16 cells (p<.001).

Mice injected with a mixture of B16 cells and LM-IL-2K^(b)/B16 cellssurvived significantly longer than mice in various control groups. Theanimals exhibited long-term resistance to the growth of the melanoma.These results demonstrate that the greatest immunotherapeutic benefitwas in the group of mice treated with immunogenic cells which expressedboth syngeneic and allogeneic MHC determinants, which were transformedwith the genomic DNA from the tumor, and which were also transformed tosecret IL-2.

The long-term immunogenic properties of LM-IL-2K^(b)/B16 cells wereinvestigated by injecting mice that survived the initial treatment witha second injection of B16 cells (FIG. 4). The second injection tookplace 150 days after the first injection of the mixture of B16 cells andLM-IL-2K^(b)/B16 cells. As a control, naive C57BL/6J mice were injecteds.c. with an equivalent number of B16 cells. There were five mice ineach group. As the data illustrate in FIG. 4, the first appearance ofmelanoma was significantly (P<.001) delayed in mice treated previouslywith LM-IL-2K^(b)/B16 cells. Mean survival time (M.S.T.) of untreatedmice was 34.4+2.2 days. M.S.T. of treated mice was 53.0+7.1 days(p<0.1).

EXAMPLE 7 Treatment of Mice having Pre-existent Melanoma withLM-IL-2K^(b)/B16 cells

To determine if LM-IL-2K^(b)/B16 cells had a similar therapeutic effecton mice with established melanomas, C57BL/6J mice were injected s.c.with 5×10³ B16 cells followed at varying times afterwards by aninjection s.c. and an injection i.p. of 2×10⁶ LM-IL-2K^(b)/B16 cells ateach injection site. An equivalent number of LM-IL-2K^(b)/B16 cells wasinjected s.c. and i.p. twice more at weekly intervals. As controls,other naive C57BL/6J mice were injected s.c. with a mixture or 5×10³ B16cells and 2×10⁶ LM-IL-2K^(b)/B16 cells, and i.p. with 2×10⁶LM-IL-2K^(b)/B16 cells, or s.c. with an equivalent number of B16 cellsalone. There were 5 mice per group. Mean survival times were as follows:mice injected with B16 cells alone, 31.8+6.1 days; mice injected with amixture of B16 cells and LM-IL-2K^(b)/B16 cells, 52.8+9.9 days; miceinjected with LM-IL-2 K^(b)/B16 cells 5 days after the injection of B16cells, 44.2+5.8 days; mice injected with LM-IL-2K^(b)/B16 cells 10 daysafter the injection of B16 cells, 39.3+3.6 days; mice injected withLM-IL-2 K^(b)/B16 cells 20 days after the injection of B16 cells,34.4+4.0 days. P for survival of mice injected with the mixture of B16cells and LM-IL-2 K^(b)/B16 cells vs. mice injected with B16 cells fivedays before LM-IL-2 K^(b)/B16 cells was less than 0.005; for miceinjected with B16 cells ten days before the injection ofLM-IL-2K^(b)/B16 cells, p was less than 0.04; for mice injected with B16cells twenty days before the injection of LM-IL-2 K^(b)/B16 cells, p wasless than 0.1.

As indicated (FIG. 5), mice injected with B16 cells five days, and tendays, before the first injection of LM-IL-2K^(b)/B16 cells survivedsignificantly longer than mice injected with B16 cells alone (p<.003 and<.04 respectively). Mice injected with B16 cells 20 days before thefirst injection of LM-IL-2K^(b)/B16 cells failed to survivesignificantly longer than mice injected only with B16 cells(M.S.T.=34.4±4 and 31.8±6 days respectively; p=0.1).

EXAMPLE 8 Antimelanoma Cytotoxic Responses in C57BL/6J Mice Immunizedwith Disrupted LM-IL-2K^(b)/B16 Cells

The experiments described above were carried out in mice immunized withviable LM-IL-2K^(b)/B16 cells. Spleen cell-mediated cytotoxicityexperiments in mice immunized with homogenized/sonicated (disrupted)LM-IL-2K^(b)/B16 cells were carried out to determine if immunizationswith disrupted cells would result in equivalent anti-melanoma cytotoxicresponses. In the experiment, 4×10⁶ LM-IL-2K^(b)/B16 cells suspended in400 μl of growth medium were homogenized in a Takmar Tissue Mixer(Cincinnati, Ohio) for one minute at 4° C. followed by sonication forone minute at 4° in a Sonifier Cell Disrupter (VWR Scientific,Philadelphia, Pa.). Afterward, naive C57BL/6J mice were injectedintraperitoneally (i.p.) and s.c. with 2×10⁶ viable or an equivalentnumber (5×10⁶) of disrupted LM-IL-2K^(b)/B16 cells at each injectionsite. The mice received two subsequent injections of the disrupted orviable cells at weekly intervals. Other naive C57BL/6J mice wereinjected according to the same protocol with viable LM-IL-2K^(b) orLM-IL-2 cells. One week after the last injection, the mice were killedand a standard ⁵¹Cr-release assay toward B16 cells was performed.

A pool of mononuclear cells from the spleens of 3 mice in each groupwere collected. A spleen cell-suspension was prepared by forcing thespleens though a number 40 gauge stainless steel screen in approximately5 ml of ice-cold growth medium. The cells were transferred to 15 mlconical centrifuge tubes (Becton Dickinson, Franklin Lakes, N.J.), andlarge clumps of cells and cell debris were allowed to settle for 1 min.Afterward, cells remaining in the supernatant were collected, overlaidonto a Histopaque 1077 gradient (Sigma) and then centrifuged (400×g) for30 min. at room temperature. The viability of the mononuclear cellscollected from the gradients at this point was greater than 90%, asdetermined by trypan blue dye exclusion (0.4%). Aliquots of thecell-suspensions were co-incubated in growth medium at 37° C. for 5 dayswith mitomycin C-treated (Sigma Chemical Co., St. Louis, Mo.) (50 ug/mlfor 45 min. at 37° C.) cells of the same type used to immunize the mice.The ratio of spleen cells to mitomycin-C-treated cells was 30:1. Theincubation medium consisted of RPMI-1640 medium supplemented with 100U/ml human IL-2, 10% FBS, 5×10⁻² mmol/L 2-β-mercaptoethanol, 15 mmol/LHEPES, 0.5 mmol/L sodium pyruvate and penicillin/streptomycin (Gibco).At the end of the 5 day incubation, the population that failed to adhereto the plastic cell culture flasks was collected and used as the sourceof effector cells for the cytotoxicity determinations.

For the ⁵¹Cr-release assay, 5×10⁶ target cells were labeled with ⁵¹Crduring a 1 hr incubation at 37° C. in growth medium containing 100 uCi⁵¹Cr (Amersham, Arlington Heights, Ill.). After three washes with DMEM,1×10⁴ of the ⁵¹Cr-labeled cells were incubated for 4 hrs. at 37° C. withthe non plastic-adherent population of spleen cells from the immunizedmice, at varying effective:target (E:T) ratios. Afterward, the percentspecific cytolysis was calculated as:$\frac{{{{Experimental}\quad}^{51}{Cr}\quad {release}} - {{{Spontaneous}\quad}^{51}{Cr}\quad {release}}}{{{{Maximum}\quad}^{51}{Cr}\quad {release}} - {{{Spontaneous}\quad}^{51}{Cr}\quad {release}}} \times 100$

Spontaneous release ranged from 10 to 15% of the maximal ⁵Cr release.

The Student t test was used to determine the statistical differencesbetween the survival and cytotoxic activities in mice in variousexperimental and control groups. A p value of less than 0.05 wasconsidered significant.

As indicated (FIG. 6), cytotoxic reactions were present in the group ofmice injected with the viable, but not the homogenized/sonicated cells.Spleen cells from mice injected with viable LM-IL-2K^(b) or LM-IL-2cells, like cells from naive mice, failed to exhibit cytotoxicity towardB16 cells.

EXAMPLE 9 Determination of the Classes of Effector Cells Activated forAnti-Melanoma Cytotoxicity in Mice Immunized with the Semi-AllogeneicTransfected Cells

The effect of anti-Lyt-2.2 monoclonal antibodies (mAbs) (directed toLyt-2.2⁺(CD8⁺T) cells) or anti-asialo G_(m1) mAbs (directed to NK/LAKcells) on spleen cell-mediated cytotoxicity reactions was used toidentify the predominant cell types activated for anti-melanomacytotoxicity in mice immunized with the semi-allogeneic transfectedcells described above.

C57BL/6J mice were injected with one of the following geneticallymodified cell-types: LM-ZipNeo, LM-IL-2, LM-IL-2/B16, LM-IL-2K^(b) andLM-IL-2K^(b)/B16. As a control, one group of mice was injected withgrowth media. The mice received 2×10⁶ cells s.c. and 2×10⁶ cells i.p.Two additional injections at weekly intervals according to the sameprotocol were also administered to the mice. Seven days after the lastinjection, a pool of mononuclear cells from the spleens of 3 mice ineach group were incubated for 5 days with mitomycin C-treated (50 ug/ml;30 min) cells from the same type as first injected. After the five dayperiod of incubation, the non-plastic-adherent cells were incubated at4° C. for 1 hour with excess quantities of the mAbs[anti-Lyt-2.2⁺(CD8⁺):hybridoma 3.155 (Sarmiento, et al. (1985) J.Immunol. 125:2665-2672) or anti-asialo G_(M1) (Kasai et al. (1980) Eur.J. Immunol. 10:175-180 (Wako Chemical Co., Dallas, Tex.)] before the⁵¹Cr-release assay was performed. The antibodies were titered such thatthe concentrations used were 5 times the amount required to saturate thebinding of the specific cell-types from naive C57BL/6 mice, asdetermined by cytofluorometric analyses of serially diluted antibodies.At the end of the incubation, ⁵¹Cr-labeled B16 cells or ⁵¹Cr-labeledc1498 cells were added and the mixed cell cultures were incubated for 4additional hours after which the specific release of isotope (%cytolysis) was determined. c1498 cells are an independently arisingneoplasm of C57BL/6 mice. Table III reflects the results of the⁵¹Cr-release assay. The values represent the mean ± SD of triplicatedeterminations.

As indicated in Table III, spleen cells from mice immunized withLM-IL-2K^(b)/B16 cells were cytotoxic for B16 cells and not c1498 cells.

As also indicated in Table III, the immune response was mediatedprimarily by CD8⁺-CTLs. NK/LAK cells did not appear to be involved inthe anti-melanoma cytotoxicity response.

EXAMPLE 10 Survival of C57BL/6J Mice Injected with a Mixture of B16Melanoma Cells and Non-Cytokine-Sec retina LMK^(b)/B16 Cells

Non-IL-2-secreting LM mouse fibroblasts (H-2^(k)) were modified toexpress H-2K^(b)-determinants (LMK^(b)) by transduction with a plasmid(pBR327H-2K^(b), Biogen Research Corp., Cambridge, Ma.), along with aplasmid (pBabePuro) conferring resistance to puromycin. The number ofpuromycin-resistant cells was expanded in vitro and then the expressionof H-2K^(b)-determinants on such cells was tested by immunofluorescentstaining essentially as described in Example 4.

After confirmation of the expression of H-2K^(b)-determinants, theLMK^(b) cells were co-transfected with genomic DNA from B16 melanomacells, along with a plasmid (pHyg) conferring resistance to hygromycin.Colonies of hygromycin-resistant, transfected cells (LMK^(b)/B16) (morethan 5×10⁴) were pooled, and the cell number was expanded in vitro. Thecells were used without further modification in the experiment.

The immunotherapeutic properties of the genetically-modified cells weretested in C57BL/6J mice (H-2^(b)). The mice, which were between 8 and 12weeks of age at the beginning of the experiment, were injectedsubcutaneously (s.c.) with a mixture of 5×10³ B16 melanoma cells and2×10⁶ LMK^(b)/B16 cells. At the same time, the mice received a secondintraperitoneal (i.p.) injection of 2×10⁶ LMK^(b)/B16 alone. The micereceived two subsequent s.c. and i.p. injections at weekly intervals ofequivalent numbers of LMK^(b)/B16 cells, without additional B16 cells.

As controls, the mice received a s.c. injection of 5×10³ B16 cellsalone, or a s.c. injection of a mixture of 5×10³ B16 cells and LM cellsmodified to express H-2K^(b)-determinants alone (LMK^(b) cells) and asecond i.p. injection of 2×10⁶ LMK^(b) cells alone. The mice in thegroup treated with LMK^(b) cells received two subsequent s.c. and i.p.injections at weekly intervals of equivalent numbers of LMK^(b) cells,without additional B16 cells.

There were eight mice in each group. P <0.001 for difference in survivalof mice injected with B16 melanoma cells and LMK^(b)/B16 cells and miceinjected with B16 cells alone, or mice injected with B16 cells andLMK^(b) cells.

As indicated in FIG. 7, mice injected with a mixture of B16 cells andLMK^(b)/B16 cells survived significantly longer than mice injected witheither B16 cells alone and mice injected with a mixture of B116 cellsand LMK^(b) cells. This result also indicates that anon-cytokine-secreting antigen presenting cell transfected with tumorgenomic DNA also provides anti-tumor immunogenic effects.

EXAMPLE 11 Formation of B16 Melanoma X LM Fibroblast Hybrid Cells

B16 melanoma X LM fibroblast hybrid cells were prepared as follows.Ouabain-resistant LM(TK-) cells, a thymidine kinase-deficient variant,were first obtained by incubating approximately 10⁷ cells for threeweeks in growth medium containing 1 mM ouabain. The medium was changedat frequent intervals, no less than every third day, to remove dead,nonadherent cells. At the end of the incubation, colonies of LM(TK-)cells (approximately 5×10²) proliferating in the ouabain-containinggrowth medium were recovered and pooled. The cells were maintained ingrowth medium containing 1 mM ouabain until use for the experiments. Forfusion, 5×10⁶ ouabain- resistant LM(TK-) cells were mixed with anequivalent number of B16 cells from in vitro culture and then incubatedat 37° in growth medium containing PEG-1000, used to facilitate fusion.Twenty four hours afterward, the medium was replaced by growth mediumcontaining both HAT and (1 mM) ouabain. LM(TK-) cells were unable togrow in HAT-containing medium due to their lack of thymidine kinase,while unfused B16 cells were able to grow in such medium. LM(TK-) cellswere selected to be ouabain-resistant, while B16 cells were sensitive toouabain. Thus the hybrid cells, resulted from the fusion of LM(TK-) andB16 cells, complemented the properties of both types of parental cellsand proliferated in a selection medium that contained both HAT andouabain.

After fusion, B16×LM hybrid cells were examined to ensure that theyexpressed MHC class I, determinants of both types of parental cells,H-2^(b) determinants from the parental B16 cells and H-2^(k)determinants from the parental LM(TK-) cells. Approximately 1×10³colonies of cells proliferating in HAT/ouabain medium were pooled andtested by quantitative immunofluorescent staining following basicallythe same protocol as described in Example 4. As indicated (FIG. 8) boththe hybrid cells, and B16 cells, stained with anti H-2K^(b) mAbs. LMcells failed to stain with anti H-2K^(b) mAbs. Under similar conditions,both the hybrid cells and LM cells stained with anti H-2K^(k) mAbs. B16cells failed to stain with anti H-2K^(k) mAbs. The intensity ofimmunofluorescent staining of the respective cell-types wasapproximately the same. The results were consistent with theco-expression by the hybrid cells of both types of MHC classI-determinants of the parental cells.

A similar approach was used to detect the formation of(antibody-defined) MAAs by the hybrid, and parental cells. An antiserumraised in C57BL/6J mice injected with killed (by three rounds offreezing and thawing) B16 cells was used for this purpose (11). Asindicated (FIG. 8), both B16 cells and the hybrid cells stained with themelanoma antibodies. TM cells failed to be stained with aliquots of themelanoma antiserum. As for MHC class I-determinants, the intensity ofimmunofluorescent staining of B16 cells and the hybrid cells with themelanoma antiserum was approximately the same. After six months ofcontinuous culture, staining of the hybrid cells with the B16 antiserumand the anti H-2k^(b) and anti H-2K^(k) mAbs was equivalent to thatfound when the cells were first investigated.

In addition to testing for the expression of MAAs and MHC classI-determinants, the hybrid cells were investigated by immunofluorescentstaining for the expression of B7.1, B7.2 and ICAM-1, co-stimulatory andadhesion molecules involved in the activation of cytotoxic Tlymphocytes. The procedure for immunostaining was essential the same asdescribed in Example 4. As indicated (FIG. 9), both the hybrid cells andLM cells stained positively with anti B7.1 mAbs (mean fluorescentintensities (MFI) of 23.2 and 19.2 above that of cells incubated withFITC-conjugated mouse IgG_(2a) alone, taken as background). LM cells andthe hybrid cells failed to stain positively for B7.2 or ICAM-1 (data notpresented). B16 cells failed to stain for B7.1, B7.2 or ICAM-1 underthese conditions (maximum MFI of 4.6 above background). WR19M.1 cells, amouse monocyte/macrophage cell line, was included as a positive control.Like the hybrid cells and LM cells, the cells stained positively withanti B7.1 mAbs (MFI of 23.2 above background).

EXAMPLE 12 Treatment with B16×LM Hybrid Cells Prolonged the Survival ofC57BL/6J Mice with Melanoma

C57BL/6J mice exhibited no apparent resistance to the malignantproliferation of B16 melanoma cells. As few as 5×10³ viable cellsinjected subcutaneously (s.c.) resulted in the death from progressivetumor growth of one hundred percent of the animals in less than 35 days.The effect of immunization with B16×LM hybrid cells on the survival ofC57BL/6J mice with B16 melanoma was determined by injecting naive mices.c. with a mixture of 5×10³ viable B16 cells and 1×10⁷ hybrid cells.The mice received a single s.c. injection of 1×10⁷ hybrid cells alonetwo weeks later. As a control, the mice received s.c. injections of anequivalent number of viable B16 cells, irradiated (5000 rads from a⁶⁰Co-source) B16 cells and UM(TK-) cells. The survival of mice in eachgroup (six per group) was compared to the survival of mice injected s.c.with 5×10³ viable B16 cells alone. As indicated (FIG. 10), mice injectedwith the mixture of B16 cells and the hybrid cells survivedsignificantly longer (P<0.005) than mice injected with the mixture ofB16 cells, irradiated B16 cells and LM(TK-) cells. The prolongedsurvival of mice injected with the hybrid cells, relative to thesurvival of mice injected with the mixture of B16 cells, irradiated B16cells and LM(TK-) cells indicated that co-expression of both H-2K^(b)and K^(k) determinants by the same cell-type was required for an optimumimmunotherapeutic result.

As additional controls, other naive C57BL/6J mice were injectedaccording to the same protocol with a mixture of B16 cells and LM cells,with a mixture of B16 cells and irradiated B16 cells or with anequivalent number of B16 cells alone. As indicated (FIG. 10), thesurvival of mice in these groups was significantly less than that ofmice in the group treated with the hybrid cells. The data indicated thatthe immunogenic properties of weak MAAs are enhanced if they wereexpressed by cells that formed both syngeneic and allogeneicdeterminants. This in vivo result was confirmed by examining the spleencell-mediated cytotoxicity in vivo. The cytotoxicity of spleen cellsobtained from C57BL/6J mice immunized with a mixture of (X-irradiated)B16 cells and LM(TK-) cells, or mice immunized with the hybrid cells wasexamined. The in vitro results were consistent with the in vivo results.

To investigate whether the hybrid cells retained the potential forgrowth in C57BL/6J mice, naive mice were injected s.c. with 10⁷ viablehybrid cells for each of three weekly injections. The mice exhibited noobvious ill effects. Tumors failed to form and the mice livedindefinitely (more than 6 months).

EXAMPLE 13 B16×LM Hybrid Cells Induced Immunity and Specific forMelanoma

To determine if mice injected with the hybrid cells developed immunitytoward other types of neoplasms originating in C57BL/6 mice, thesurvival of naive C57BL/6J mice injected with a mixture of the hybridcells and B16 cells was compared with the survival of mice injected witha mixture of the hybrid cells and Gl 261 glioma, c1498 lymphoma cells orEL4 thymoma cells. The animals (five per group) received 5×10³ of therespective tumor cells and 1×10⁷ hybrid cells at the first injection,and 1×10⁷ hybrid cells alone at weekly intervals on two subsequentoccasions.

As indicated (FIG. 11), mice injected with the mixture of B16 cells andhybrid cells survived significantly (p<0.005) longer than mice in any ofthe other groups. With the exception of mice injected with B16 cells andthe hybrid cells, mice in the control groups failed to survive longerthan mice injected with an equivalent number of the respective tumorcells alone. The results were consistent with the expression ofmelanoma-specific, tumor associated antigens (TAAs) in a highlyimmunogenic form by the melanoma X fibroblast hybrid cells.

Spleen cell-mediated cytotoxic reactions in mice injected with thehybrid cells were carried-out to determine if the specificity of theanti-melanoma immune-responses found in vivo was reflected by theresults of studies performed in vitro. Naive C57BL/6J mice (two pergroup) were injected s.c. three times at weekly intervals with 1×10⁷viable hybrid cells. A spleen cell-suspension, prepared three weeksafter the last injection, was tested for its reactivity toward⁵¹Cr-labeled B16 cells, and, for comparison, against ⁵¹Cr-labeled c1498,EL-4 or Gl 261 cells as well. The results (FIG. 12) indicated that thereactivity toward the melanoma cells was significantly (p<0.0005) higherthan the reactions toward any of the other the other types of murinetumors tested. The reactivity toward c1498, EL-4 or Gl 261 in miceimmunized with the hybrid cells was not greater than that of naive mice.

EXAMPLE 14 CD8⁺ (Lyt 2.2) Cells were the Predominant Anti MelanomaEffector Cell-type in Mice Immunized with B16×LM Hybrid Cells

The effect of mAbs for CD8⁺ (Lyt 2.2), CD4⁺ (L3T4) or asialoGM1-determinants on effector cells mediating the anti B16 melanomaresponse was used to determine the types of cells activated for antimelanoma immunity in mice immunized with the hybrid cells. NaiveC57BL/6J mice (two per group) were injected s.c. with 1×10⁷ hybrid cellsfor three weekly injections. One week after the last injection, the micewere killed and a spleen cell suspension was prepared. The cells wereincubated in growth medium for 5 days additional days with mitomycinC-treated, (50 ug/ml; 30 min.; 37° C.) hybrid cells (ratio of hybridcells : spleen cells=30:1). At the end of the incubation, the spleencells were treated with excess quantities of CD4⁺, CD8⁺, and/or asialoGM1 mAbs for 1 hr. at 4° C. before a standard ⁵¹Cr release assay towardB16 cells was performed. The procedure is as described in Example 9.

As indicated (FIG. 13), treatment of the cells with anti-CD8⁺ mAbs orwith a mixture of CD8⁺ and asialo GM1 mAbs reduced the specific releaseof isotope to “background” (the anti melanoma activity present in apopulation of spleen cells from naive C57BL/6J mice) (p<0.005). Lesserinhibitory effects were detected in cell populations treated with CD4⁺or asialo GM1 mAbs alone. These results indicate that CD8+ cells are thepredominant type of anti-melanoma effector cells in mice immunized withthe B16×LM hybrid cells.

EXAMPLE 15 Survival of C57BL/6J Mice Injected with a Mixture of E0771Breast Carcinoma Cells and LM-IL-2K^(b) Cells Transfected with DNA fromE0771 Cells (LM-IL-2K^(b)/EO771)

EO771 is a breast cancer cell line derived from a breast neoplasm thatarose in a C57BL/6 mouse. The cells are maintained by serial transfer insyngeneic mice, or under standard cell culture conditions. C57BL/6J miceare highly susceptible to EO771 cells. One hundred percent of miceinjected with 1×10³ EO771 cells developed progressively growingneoplasms.

The potential immunotherapeutic properties of LM-IL-2K^(b)/EO771 cellsagainst the growth of EO771 cells were determined in naive syngeneicC57BL/6J mice (FIG. 14). In the experiment, C57BL/6J mice (7 per group)were injected into the fat pad of the breast with a mixture of 5×10³EO771 cells and 2×10⁶ LM-IL-2K^(b)/EO771 cells in a total volume of 200μl. At the same time the mice also received an i.p. injection of 2×10⁶LM-IL-2K^(b)/EO771 cells alone, followed by two subsequent injections atweekly intervals of 2×10⁶ LM-IL-2K^(b)/EO771 cells i.p. and 2×10⁶LM-IL-2K^(b)/EO771 cells into the same breast as first injected, withoutadditional EO771 cells. As a control, naive C57BL/6J mice were injectedinto the breast with an equivalent number of EO771 cells in growth mediaalone, followed by two subsequent injections at weekly intervals ofgrowth media i.p. and growth media into the same breast as firstinjected. As additional controls, naive C57BL/6J mice were injectedaccording to the same protocol with a mixture of EO771 cells and LMcells, with EO771 cells and non tumor-DNA transfected LM-IL-2K^(b)cells, or with EO771 cells and LM-IL-2K^(b) cells transfected with DNAfrom B16 cells (LM-IL-2K^(b)/B16). B16 is a melanoma cell line ofC57BL/6J origin.

The results (FIG. 14) indicate that the first appearance of tumor wasdelayed in the group of mice injected with the mixture of EO771 cellsand LM-IL-2K^(b)/EO771 cells, relative to that of mice in any of theother groups. Three mice in the group receiving EO771 cells andLM-IL-2K^(b)/EO771 cells failed to develop tumors. In those instances inwhich breast neoplasms appeared, the rate of tumor growth (twodimensional measurements) in each group was the same. These resultsindicated that specific partial immunity toward EO7771 cells developedin mice immunized with LM-IL-2K^(b)/EO771 cells.

The development of partial immunity in C57BL/6J mice treated withLM-IL-2K^(b)/EO771 cells was emphasized by the finding that mice in thetreatment group survived significantly (P<0.01) longer than mice in anyof the various control groups, including mice injected with the mixtureof EO771 cells and LM-IL-2K^(b)/EO771 cells (FIG. 15). In someinstances, mice injected with the mixture of EO771 cells andLM-IL-2K^(b)/EO771 cells appeared to have rejected the breast cancercells and survived indefinitely, more than 110 days. In addition, tumorsfailed to form in mice injected with semi-allogeneic LM-IL-2K^(b)/EO771cells alone, or within the peritoneal cavities of mice injected into thebreast with the mixture of EO771 cells and LM-IL-2K^(b)/EO771 cells.

To determine if surviving mice in the group injected with EO771 cellsand LM-IL-2k^(b)/EO771 cells were resistant to a second injection ofEO771 cells, the animals were injected into the fat pad of the breastwith 5×10³ EO771 cells alone 110 days after the first injection. The MST(33±6 days) of the surviving mice was significantly (P<0.02) greaterthan that of naive mice injected into the breast with an equivalentnumber of EO771 cells alone (20±6 days) (FIG. 16).

EXAMPLE 16 Survival of C3H/HeJ Mice Injected with a Mixture of Cellsfrom a Spontaneous Adenocarcinoma of the Breast (SB-1) and LM-IL-2K^(b)Cells Transfected with DNA from the same Neoplasm (LM-IL-2K^(b)/SB-1)

The results of the prior experiments (Example 15) indicated thatspecific, partial immunity toward EO771 cells, a breast cancer cellline, was generated in C57BL/6J mice immunized with semi-allogeneic,IL-2-secreting mouse fibroblasts transfected with DNA from EO771 cells.Since the immunogenic properties of a breast cancer cell line mightdiffer from those of a spontaneous breast neoplasm, the same protocolwas followed to determine if an analogous response would be observed inC3H/HeJ mice immunized with semi-allogeneic, IL-2-secreting mousefibroblasts transfected with DNA taken directly from a breastadenocarcinoma arising in a C3H/He mouse (SB-1 cells). Untreated C3H/HeJmice exhibited no apparent resistance to the growth of SB-1 breastcarcinoma cells. One hundred percent of mice injected into the breastfat pad with 1×10⁴ SB-1 cells died from progressive tumor growth inapproximately 30 days. The potential immunotherapeutic properties ofLM-IL-2K^(b)/SB-1 cells were determined by injecting C3H/HeJ mice intothe fat pad of the breast with a mixture of 1×10⁶ SB-1 cells and 2×10⁶LM-IL-2K^(b)/SB-1 cells, and i.p. with 2×10⁶ LM-IL-2K^(b)/SB-1 cellsalone. The mice received two subsequent injections i.p. and twosubsequent injections into the same breast as first injected with thesame number of LM-IL-2K^(b)/SB-1 cells alone, as described previously.The time to the first appearance of tumor, rate of tumor growth andsurvival of mice injected with the mixture of SB-1 cells andLM-IL-2K^(b)/SB-1 cells was compared to the time to first appearance andsurvival of mice injected with SB-1 cells alone. There were five miceper group. The results (FIG. 17) indicated that the first appearance ofa palpable tumor in the breasts of mice injected with the mixture ofLM-IL-2K^(b)/SB-1 cells and SB-1 cells was delayed, relative to thefirst appearance of tumor in mice injected with SB-1 cells and growthmedia. Once the breast neoplasms first appeared, the rate of tumorgrowth (two dimensional measurements) in the treated and untreatedgroups was approximately the same. Consistent with the delayedappearance of tumor in the treated group, mice injected with the mixtureof SB-1 cells and LM-IL-2K^(b)/SB-1 cells survived significantly(P<0.006) longer than mice injected with SB-1 cells alone (FIG. 18). Inno instances were tumors detected at immunization sites injected withLM-IL-2K^(b)/SB-1 cells alone.

As additional controls, naive C3H/HeJ mice were injected according tothe same protocol with a mixture of SB-1 cells and non transfectedLM-IL-2 cells, with SB-1 cells and non transfected semi-allogeneicLM-IL-2K^(b) cells, or with SB-1 cells and syngeneic LM-IL-2 cellstransfected with DNA from SB-1 cells (LM-IL-2/SB-1). As indicated (FIGS.17 and 18), with the exception of two mice in the group (5 per group)injected with the mixture of SB-1 cells and LM-IL-2K^(b)/EO771 cells,the first appearance of tumor, rate of tumor growth and survival of micein each group was approximately the same as that of mice injected withSB-1 cells alone. The greatest immunotherapeutic benefit was in thegroup of mice injected with the mixture of SB-1 cells and LM-IL-2K^(b)cells transfected with genomic DNA from SB-1 cells.

As an additional control, to determine the effect of immunizations withLM-IL-2K^(b)/EO771 cells on the growth of SB-1 cells, the independentlyarising breast neoplasm, naive C3H/HeJ mice were injected with a mixtureof SB-1 cells and LM-IL-2K^(b)/EO771 cells. As indicated (FIG. 18),although mice injected with the mixture of SB-1 cells andLM-IL-2K^(b)/EO771 cells survived longer than mice injected with SB-1cells alone, they died in significantly (P<0.01) shorter intervals thanmice injected with SB-1 cells and LM-IL-2K^(b) cells transfected withDNA from SB-1 cells.

EXAMPLE 17 Spleen Cell-mediated Immune Responses toward EO771 Cells wereGenerated in C57BL/6J Mice Immunized with LM-IL-2K^(b)/EO771 Cells

As described in Example 15, C57BL/6J mice injected with a mixture ofEO771 cells and LM-IL-2K^(b)/EO771 cells survived significantly longerthan mice in various control groups, including mice injected with amixture of EO771 cells and LM-IL-2K^(b) transfected with DNA from B16melanoma cells. The results indicate that specific, partial immunitytoward EO771 cells was generated in mice immunized with thesemi-allogeneic, cytokine-secreting cells transfected with DNA fromEO771 cells.

A standard ⁵¹Cr-release assay was used to characterize the anti-tumorimmune response in mice injected with the mixture of EO771 cells andLM-IL-2K^(b)/EO771 cells. In the experiment, C57BL/6J mice were injectedinto the fat pad of the breast with a mixture of 5×10³ EO771 cells and2×10⁶ LM-IL-2K^(b)/EO771 cells. At the same time the mice also receivedan injection i.p. of equivalent numbers of LM-IL-2K^(b)/EO771 cellsalone, followed by two subsequent injections at weekly intervals ofequivalent numbers of LM-IL-2K^(b)/EO771 cells i.p. andLM-IL-2K^(b)/EO771 cells into the same breast as first injected, withoutadditional EO771 cells. The mice were sacrificed one week after the lastinjection of LM-IL-2K^(b)/EO771 cells. A pool of mononuclear cells fromthe spleens of 3 mice in each group were collected. A spleencell-suspension was prepared and coincubated for five additional dayswith (mitomycin C-treated; 50 ug/ml; 30 min. at 370) LM-IL-2K^(b)/EO771cells, after which a cytotoxicity determination toward ⁵¹Cr-labeledEO771 cells was performed. As controls, same protocol was followedexcept that spleen cells from mice injected with EO771 cells and LMcells, EO771 cells and LM-IL-2K^(b) cells, or EO771 cells andLM-IL-2K^(b)/B16 cells were substituted for spleen cells from miceinjected with EO771 cells and LM-IL-2K^(b)/EO771 cells. As an additionalcontrol, spleen cells were obtained from mice injected with EO771 cellsalone.

The results (Table IV) indicate that the cytotoxic responses (specific⁵¹Cr-release) toward EO771 cells in mice injected with the mixture ofEO771 cells and LM-IL-2K^(b)/EO771 cells were significantly (P<0.01)higher than those in any of the other groups. The response toward EO771cells in mice injected with the mixture of EO771 cells and LM-IL-2k^(b)cells transfected with DNA from B16 melanoma cells was not significantlydifferent than background (specific ⁵¹Cr-release from EO771 cellsco-incubated with spleen cells from mice injected with EO771 cellsalone). Immunizations with non-DNA-transfected LM-IL-2K^(b) cells failedto generate cell mediated responses toward EO771 breast carcinoma cellsin C57BL/6J mice.

As an additional control, to determine if cytotoxic responses toward LMcells were present in the spleen cell-suspensions that failed to reactwith EO771 cells, aliquots of the cell-suspensions from C57BL/6J miceinjected with the different cell mixtures were tested for cytotoxicresponses toward LM cells. As indicated (Table IV), the percent specificlysis was greater than fifty percent for cells from each group includingcells from mice immunized with LM-IL-2K^(b)/B16 cells that failed togenerate cytotoxic responses toward EO771 cells.

EXAMPLE 18 CD8+ Cells Infiltrated Breast Tumors Developing in MiceInjected with SB-1 and LM-IL-2K^(b)/SB-1 Cells and Mice Infected withEO771 and LM-IL-2K^(b)/EO771 Cells

As described in Example 16, C3H/HeJ mice injected with a mixture of SB-1cells and LM-IL-2K^(b)/SB-1 cells survived significantly longer thanmice in various control groups.

Immunihistochemical staining was used to characterize the cellularinfiltrate in breast tumors developing in mice injected with the mixtureof SB-1 cells and LM-IL-2K^(b)/SB-1 cells. Primary antibodies for mouseCD4(L3T4), CD8a (Ly-2), CD11b or NK (Ly-49c) cells were used in theanalysis. In these experiments, C3H/HeJ mice (3 mice per group) wereinjected into the fat pad of the breast with a mixture of 1×10⁶ SB-1cells and 2×10⁶ LM-IL-2K^(b)/SB-1 cells, and i.p. with 1×10⁶LM-IL-2K^(b)/SB-1 cells alone. The mice received two subsequent i.p.injections and two subsequent injections into the same breast as firstinjected with equivalent numbers of LM-IL-2K^(b)/SB-1 cells alone, asdescribed previously. One week after the last injection, the mice weresacrificed and breast neoplasms were quickly frozen in liquid nitrogen.

A representative tissue block was selected and 5-um frozen sections wereprepared, mounted on clean glass slides and fixed with acetone.Afterwards, the sections were washed two times with 0.1 M PBS, placed in3% H₂O₂ for 10 mins., and then washed three times with 0.1 M PBS. A 1:5dilution of goat serum (Gibco BRL) in PBS (blocking buffer) was added tothe slides (to reduce non-specific binding of primary antibodies)followed by incubation at 37° for 15 mins. After incubation, the slideswere flooded with anti-mouse CD4 (L3T4), anti-mouse CD8a (Ly-2),anti-mouse CD11b (integrin a_(m) Mac-1a) chain or anti-mouse NK (Ly-49c)antibdies (all from Pharmingen, San Diego, Calif.). These antibodies hadbeen titrated such that the dilution used gave the minimum backgroudstaining, typically a 1:50 dilution with blocking buffer. Afterwards,the slides were washed two times with PBS followed by the addition ofavidin-biotin complex and diaminobenzidine, according to themanufcture's instructions (Vector, Burlington, Calif.). The sectionswere washed two times with PBS and counterstained with eosin. After afinal wash with xylene, coverslips were placed over the stained sectionsand mounted with Permount. The distribution of cells that stained withthe mAbs was evaluated independently by four investigators and gradedquantitatively.

As indicated (FIG. 19 and Table V), large numbers of cells reactive withCD8 antibodies infiltrated the epithelial ducts of the breast tumors inmice injected with the mixture of SB-1 and LM-IL-2K^(b)/SB-1 cells.Lesser numbers of CD8+ cells were present in tumors in mice injectedwith SB-1 cells alone. There were no apparent differences between thenumbers of CD4+, CD11b+ or NK cells in breast neoplasms of the treatedand untreated groups (Table V).

The same protocol was then followed to charaterize the cellularinfiltrates in epithelial ducts of tumors forming in C57BL/6J miceinjected with a mixture of EO771 cells and LM-IL-2K^(b)/EO771 cells.Similar CD8+ T cell-infiltrates were present in breast tumors developingin C57BL/6J mice injected with a mixture of EO771 cells andLM-IL-2K^(b)/EO771 cells. Lesser numbers of CD8+ cells were present intumors developing in mice injected with EO771 cells alone.

TABLE I CURRENTLY RECOGNIZED HLA SPECIFICITIES DETECTED AT EACH HLASUBREGION (ROITT ET AL.) DR DO DP B C A DR1 Dw1 DQw1 DPw1 Bw4 Bw47 Cw1A1 DR2 Dw2 DQw2 DPw2 B5 Bw48 Cw2 A2 DR3 Dw3 DQw3 DPw3 Bw6 B49 Cw3 A3 DR4Dw4 DPw4 B7 Bw50 Cw4 A9 DR5 DPw5 B8 B51 Cw5 A10 DRw6 DPw6 B12 Bw52 Cw6A11 DR7 Dw7 B13 Bw53 Cw7 Aw19 DRw8 Dw8 B14 Bw54 Cw8 A23 DRw9 B15 Bw55A24 DRw10 B16 Bw56 A25 DRw11 Dw5 B17 Bw57 A26 DRw12 B18 Bw58 A28 DRw13Dw6 B21 Bw59 A29 DRw14 Dw9 Bw22 Bw60 A30 DRw52 B27 Bw61 A31 DRw53 B35Bw62 A32 B37 Bw63 Aw33 B38 Bw64 Aw34 B39 Bw65 Aw36 B40 Bw67 Aw43 Bw41Bw70 Aw66 Bw42 Bw71 Aw68 B44 Bw72 Aw69 B45 Bw73 B46

TABLE II INTERLEUKIN-2 SECRETION BY GENETICALLY MODIFIED FIBROBLASTSVACCINE IL-2^(a) cell type (units/10⁶ cells/48 Hr) LM- ZipNeo  0 LM-IL-296 LM-IL-2/B16 98 LM-IL-2K^(b) 91 LM-IL-2K^(b)/B16 86

TABLE III CYTOTOXICITY OF B16 BY SPLEEN CELLS FROM MICE VACCINATED WITHSEMI-ALLOGENEIC FIBROBLASTS TRANSFECTED WITH GENOMIC DNA FROM B16SPECIFIC VACCINE CELL TYPE TARGET AB BLOCKING RELEASE Media B16 none 0.0B16 α CD8⁺ 0.0 B16 α Asialo-GM1 0.0 c1498 none 0.0 LM-ZipNeo B16 none4.9 ± 1.5 B16 α CD8⁺ 3.6 ± 1.8 B16 α Asialo-GM1 1.2 ± .95 c1498 none 2.2± .62 LM-IL-2 B16 none 0.0 B16 α CD8⁺ 0.0 B16 α Asialo-GM1 0.0 c1498none 0.0 LM-IL-2/B16* B16 none 2.0 ± .3 B16 α CD8⁺ 3.3 ± .35 B16 αAsialo-GM1 1.4 ± 1.4 c1498 none 3.1 ± 2.1 LM-IL-2K^(b) B16 none 6.3 ±2.1 B16 α CD8⁺ 2.0 ± 1.7 B16 α Asialo-GM1 3.3 ± .75 c1498 none 4.6 ± 2.4LM-IL-2K^(b)/B16** B16 none 19.1 ± .36 B16 α CD8⁺ 9.3 ± 2.0 B16 αAsialo-GM1 17.6 ± 2.7 c1498 none 3.8 ± 1.6

TABLE IV CYTOTOXIC RESPONSES TOWARD EO771 BREAST CARCINOMA CELLS INC57BL/6J MICE INJECTED WITH A MIXTURE OF EO771 CELLS AND LM-IL-2K^(B)/EO771 CELLS Injected with EO771 cells Target % specific ⁵¹Cr-releaseand EO771 25.0 ± 7 LM-IL-2K^(b)/EO771 cells LM cells EO771 9.0 ± 4LM-IL-2K^(b) cells EO771 3.1 ± 2.0 LM-IL-2K^(b)/B16 cells EO771 7.0 ±4.0 Media EO771 3.3 ± 1.0 EO771 cells and LM-IL-2K^(b)/EO771 cells LM 59± 12 LM cells LM 64 ± 15 LM-IL-2K^(b) cells LM 53 ± 3 LM-IL-2K^(b)/B16cells LM 57 ± 10 Media LM 1.8 ± 12 Legend to Table IV: C57BL/6J micewere injected into the fat pad of the breast with a mixture of 5 × 10³EO771 cells and 2 × 10⁶ LM-IL-2K^(b)/EO771 cells or with equivalentnumbers of EO771 cells and LM cells, EO771 cells and LM-IL-2K^(b) cells,with EO771 cells and LM-IL-2K^(b)/B16 cells, or with EO771 cells ingrowth media. The mice received two subsequent injections i.p. and intothe breast of equivalent numbers of LM # cells, LM-IL-2K^(b) cell, orLM-IL-2K^(b)/B16 cells, without additional EO771 cells. One week afterthe last injection, the mice were killed, and pooled spleencell-suspensions from mice in each group were mixed with mitomycinC-treated (50 ug/ml for 30 min. at 37° C.) stimulator cells of the sametype used to immunize the mice, followed by incubation at 37° C. understandard cell culture conditions for five days. At the end of theincubation, a # ⁵¹Cr-release assay was performed, using ⁵¹Cr-labeledEO771 cells or ⁵¹Cr-labeled LM cells as “targets” the reaction. Theratio of spleen cells to target cells was 100:1.

TABLE V IMMUNOHISTOCHEMICAL STAINING OF BREAST NEOPLASMS IN MICEINJECTED WITH SB-1 CELLS AND LM-IL-2K^(B)/SB-1 CELLS Infiltrating CellsCD4 CD8 CD11b NK Injected with SB-1 1.1 ± 0.9 9.9 ± 3.4 6.5 ± 3.0 0.4 ±0.5 and LM-IL-2K^(b)/SB-1 cells Injected with SB-1 2.0 ± 1.6 0.9 ± 1.48.0 ± 2.0 <0.1 ± 0.1  cells alone Legend: C3H/HeJ mice were injectedinto the fat pad of the breast with a mixture of 1 × 10⁶ SB-1 cells and2 × 10⁶ LM-IL-2K^(b)/SB-1 cells in a total volume of 200 μl. At the sametime the mice received an injection i.p. of 2 × 10⁶ LM-IL-2K^(b)/SB-1cells in 200 μl alone, followed by two subsequent injections at weeklyintervals of 2 × 10⁶ LM-IL-2K^(b)/SB-1 cells i.p. # and 2 × 10⁶LM-IL-2K^(b)/SB-1 cells into the fat pad of the same breast as firstinjected. As controls, other naive C3H/He mice were injected accordingto the same protocol with equivalent numbers of SB-1 cells into thebreast alone, without subsequent injections. One week after the lastinjection, histologic sections were prepared for immunohistochemicalstaining with CD4, CD8, CD11b or NK mAbs. The data represent an #examination of cell numbers in five high powered fields per each ofeight slides by three independent observers. P < .001 for difference innumber of CD8⁺ cells in tumors of mice injected with SB-1 cells andLM-IL-2K^(b)/SB-1 cells and mice injected with SB-1 cells alone. P fordifference in number of CD4⁺, CD11b or NK cells in tumors of miceinjected with SB-1 cells and LM-IL-2K^(b)/SB-1 cells and mice injectedwith SB-1 cells alone, not significant.

What is claimed is:
 1. A semi-allogeneic immunogenic cell foradministration to an animal recipient, which comprises anantigen-presenting cell expressing at least one class I MHC or class IIMHC determinant that is syngeneic to the recipient and at least oneclass I or class II MHC determinant that is allogeneic to the recipient,wherein said antigen presenting cell is transformed with and expressesDNA coding for at least one antigen, and wherein said antigen or a partthereof, when complexed with said MHC class I or class II determinant atthe cell surface, is recognized by T cells.
 2. A semi-allogeneicimmunogenic cell for administration to an animal recipient, whichcomprises an antigen-presenting cell expressing at least one class I MHCor class II MHC determinant that is syngeneic to the recipient and atleast one class I or class II MHC determinant that is allogeneic to therecipient and wherein said antigen presenting cell is transformed withand expresses DNA isolated from a neoplasm or a tumor of the recipient.3. The semi-allogeneic immunogenic cell of claim 1 or 2, wherein saidantigen presenting cell is further transformed with a coding sequencefor at least one cytokine.
 4. The semi-allogeneic immunogenic cell ofclaim 3 wherein the cytokine is selected from the group consisting ofinterleukin-1, interleukin-2, interleukin-3, interleukin-4,interleukin-5, interleukin-6, interleukin-7, interleukin-8,interleukin-9, interleukin-10, interleukin-11, interleukin-12,interferon-α, interferon-γ, tumor necrosis factor, granulocytemacrophage colony stimulating factor, and granulocyte colony stimulatingfactor.
 5. The semi-allogeneic immunogenic cell of claim 1 or 2, whereinthe antigen-presenting cell is selected from the group consisting of afibroblast, a macrophage, a B cell, and a dendritic cell.
 6. Thesemi-allogeneic immunogenic cell of claim 2, wherein the neoplasm isselected from the group consisting of melanoma, lymphoma, plasmocytoma,sarcoma, glioma, thymoma, leukemias, breast cancer, prostate cancer,colon cancer, esophageal cancer, brain cancer, lung cancer, ovarycancer, cervical cancer, and hepatoma.
 7. The semi-allogeneicimmunogenic cell of claim 2 wherein the DNA isolated from a neoplasm ortumor comprises coding sequences for tumor associated antigens.
 8. Thesemi-allogeneic immunogenic cell of claim 2 wherein the DNA isolatedfrom neoplastic cells comprises coding sequences for tumor associatedantigens that are associated with a tumor, wherein said tumor isselected from the group consisting of melanoma, lymphoma, plasmocytoma,sarcoma, glioma, thymoma, leukemias, breast cancer, prostate cancer,colon cancer, esophageal cancer, brain cancer, lung cancer, ovarycancer, cervical cancer, and hepatoma.
 9. A therapeutic compositioncomprising the semi-allogeneic immunogenic cell of at least one ofclaims 1, 2, 7, or 8 admixed with a therapeutically acceptable carrier.10. A therapeutic composition comprising the semi-allogeneic immunogeniccell of claim 3 admixed with a therapeutically acceptable carrier.
 11. Atherapeutic composition comprising the semi-allogeneic immunogenic cellof claim 4 admixed with a therapeutically acceptable carrier.
 12. Atherapeutic composition comprising the semi-allogeneic immunogenic cellof claim 5 admixed with a therapeutically acceptable carrier.
 13. Atherapeutic composition comprising the semi-allogeneic immunogenic cellof claim 6 admixed with a therapeutically acceptable carrier.
 14. Asemi-allogeneic immunogenic cell for administration to an animalrecipient, which comprises an antigen-presenting cell expressing atleast one of class I or class II MHC determinants, wherein said antigenpresenting cell is genetically selected such that at least one of saidclass I MHC or class II MHC determinants is syngeneic to the recipientand at least one of said class I or class II MHC determinants isallogeneic to the recipient, wherein said antigen presenting cellexpresses at least one antigen, and wherein said antigen or a partthereof, when complexed with said MHC class I or class II determinant atthe cell surface, is recognized by T cells.